Identification of a CTX-M-255 β-lactamase containing a G239S substitution selectively conferring resistance to penicillin/β-lactamase inhibitor combinations

Abstract

Objectives

An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized β-lactamase, CTX-M-255, as the only acquired β-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported β-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15.

Methods

All β-lactamase genes were expressed in E. coli TOP10 and MICs to representative β-lactam-antibiotics were determined. Furthermore, bla_CTX-M-15,  bla_CTX-M-27, bla_CTX-M-178 and bla_CTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis–Menten kinetic parameters against representative β-lactam-antibiotics and IC₅₀s of clavulanate, sulbactam, tazobactam and avibactam.

Results

TOP10-transformants expressing bla_CTX-M-178 and bla_CTX-M-255 showed resistance to penicillin/β-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing bla_CTX-M-15 and bla_CTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC₅₀s of β-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255.

Conclusions

CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M β-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.

Introduction

Penicillin/β-lactamase-inhibitor combinations (P/BLI) constitute an important class of antibiotics in medicine and include amoxicillin/clavulanate, ampicillin/sulbactam and piperacillin/tazobactam. In E. coli resistance to P/BLIs with accompanying decreased susceptibility to cephalosporins may result from increased expression of endogenous Class C (AmpC) enzymes or the acquisition of ESBL or AmpC enzymes. Common resistance mechanisms identified in cephalosporin-susceptible E. coli resistant to amoxicillin/clavulanate include the expression of OXA-1 β-lactamase, hyperproduction of TEM-1 β-lactamase and presence of inhibitor-resistant TEM-variants (IRT).¹ Clinical resistance to amoxicillin/clavulanate seems to occur more readily than to piperacillin/tazobactam, but resistance to piperacillin/tazobactam has been attributed to all three mechanisms.^2,3

In contrast to inhibitor-resistant enzymes in the TEM series, inhibitor resistance of enzymes of the CTX-M series has only been infrequently reported.⁴ Two enzymes, CTX-M-190 and CTX-M-199, both containing an S133T substitution, have been described in detail.^5,6 Both enzymes retained hydrolytic activity against penicillins and oxyimino-cephalosporins, but acquired resistance to inhibition by sulbactam, tazobactam and avibactam, but not clavulanate. Sequences of additional CTX-M-enzymes, CTX-M-218 (NCBI accession number NG057613) and CTX-M-234 (NG068168), containing an S133T substitution, have been deposited, but the enzymes have not been characterized. In an in vitro selection study using amoxicillin-clavulanate selection, Ripoll et al. derived CTX-M-variants containing a S133G substitution.⁷ These displayed resistance to P/BLI-combinations and lost activity against oxyimino-cephalosporins. In another in vitro selection experiment using piperacillin/tazobactam, Rosenkilde et al. isolated CTX-M-15 variants with S133G (also identified in a clinical isolate as CTX-M-189; NG051468) or G239S substitutions (also identified in a clinical isolate as CTX-M-178; NG056408) showing decreased susceptibility to piperacillin/tazobactam with a concomitant loss of activity against oxyimino-cephalosporins.⁸ Despite their detection in clinical isolates and their identification in in vitro evolution studies for P/BLI resistance these CTX-M-variants have remained largely uncharacterized.

As part of a survey of resistance mechanisms underlying piperacillin/tazobactam resistance, but susceptibility to oxyimino-cephalosporins (e.g. cefuroxime, cefotaxime and ceftazidime) in E. coli, we identified E. coli WGS1363 containing a previously undescribed variant of CTX-M-27 containing a G239S substitution.⁹ Here we characterize the isolate and its β-lactamase CTX-M-255 (NG081696) and compare it to CTX-M-178.

Materials and methods

Study isolate

E. coli WGS1363 was isolated in 2018 from a urine culture in the Department of Clinical Microbiology, Copenhagen University Hospital—Amager and Hvidovre, Denmark, and was part of a collection of piperacillin/tazobactam-resistant, but oxyimino-cephalosporin-susceptible isolates.⁹

Whole-genome sequencing

Whole-genome sequencing of E. coli WGS1363 was done as previously described using both Illumina and Oxford Nanopore Technology sequencing methods. Hybrid assembly of the resulting sequencing reads was made using Unicycler.¹⁰ Sequences were analysed using ResFinder v.4.1, SeroTypeFinder v.2.0, FimTyper v.1.0 and PlasmidFinder v.2.1 (https://www.genomicepidemiology.org/services/).^11–14 Sequence data are available at NCBI BioProject no. PRJNA855633.

Antimicrobial susceptibility testing

MICs were determined in triplicate determinations using broth microdilution following EUCAST methodology, except for mecillinam for which agar dilution was used. Median MIC is reported. E. coli ATCC 25922 and ATCC 35218 were used as quality control strains as appropriate. All MIC determinations with controls strains were within the EUCAST range.

β-lactamase activity determination

β-lactamase activity of WGS1363 was determined using the Amplite^™ Colorimetric β-lactamase Activity Assay Kit (AAT Bioquest) as previously described.³

Construction of E. coli TOP10/pZS3(*) transformants

Plasmids pZS3-CTX-M-15 and pZS3-CTX-M-178 were previously described.⁸ Briefly, these low-copy plasmids express CTX-M-15 and CTX-M-178 under the control of a bla_TEM  P3 promoter and have a bla_TEM terminator downstream of the β-lactamase genes. To allow comparison to the parental isolate, WGS1363, we constructed pZS3*-CTX-M-255 and pZS3*-CTX-M-27 transformants in the same low-copy plasmid but with a native ISEcp1-derived promoter identical to the promoter controlling bla_CTX-M-255 in WGS1363. Details of the protocol are provided as Supplementary Information (available as Supplementary data at JAC Online).

Enzyme kinetics and inhibition assays

Construction and expression of recombinant proteins were performed as described.^15,16 Briefly, the genes encoding β-lactamases, CTX-M-15, CTX-M-27, CTX-M-178 and CTX-M-255 with a C-terminal 6xHis tag were cloned into the expression vector pET24c using isothermal Gibson assembly (New England Biolabs), and transformed into E. coli BL21 STAR (New England Biolabs). Expressed protein was affinity purified on a column containing NiNTA beads (Profinity^™ IMAC Resin, Ni-charged, Bio-Rad) and eluted with imidazole. Purity of the proteins was verified using SDS–PAGE. Proteins were stored at 4°C until use.

The degradation of β-lactams was measured using a Shimadzu UV-2600 UV-visible light spectrophotometer by monitoring the specific absorption of the substrates (nitrocefin, 380 nm; penicillin G, 235 nm; piperacillin, 235 nm; ampicillin, 235 nm; cephalothin, 273 nm, cefotaxime, 260 nm, meropenem, 304 nm). For nitrocefin product formation was measured at 500 nm. Measurements were done at room temperature using a final enzyme concentration of 1 nM for CTX-M-15 and CTX-M-27, and 100 nM for CTX-M-255 and CTX-M-178.

To determine the inhibitory concentrations for β-lactamases-inhibitors, we monitored the formation of the degradation product of 100 µM nitrocefin for 10 min using a spectrophotometer. We normalized the A_500nm value after 10 min by setting the sample without inhibitor as 100% remaining activity and the fully inhibited sample (highest inhibitor concentration) as 0% remaining activity. The remaining activity was then plotted against the inhibitor concentration, and the IC₅₀ values were calculated using an inhibitor versus response function.

Data analysis was done using GraphPad Prism (v.8.3.0).

Protocols are further detailed in the Supplementary Information.

Results and discussion

Sequencing of WGS1363 showed that the isolate belonged to the ST131 C1 clade, was serotype O25:H4 and contained the fimH30 allele. It contained a previously undescribed bla_CTX-M-255 as the only acquired β-lactamase gene. The nucleotide sequence of bla_CTX-M-255 differed from bla_CTX-M-27 only at position 715 resulting in a G239S amino acid substitution. Hybrid assembly of Illumina and Oxford Nanopore Technology sequences showed that bla_CTX-M-255 was present on a 106 723 bp F1:A2:B20 plasmid. bla_CTX-M-27-containing F1:A2:B20 plasmids have been strongly associated with the global E. coli ST131 O25:H4 H30R1/C1-M27 clade.¹⁷ As reported for bla_CTX-M-27 in F1:A2:B20 plasmids, a truncated ISEcp1 element is located 42 bp upstream of bla_CTX-M-255.¹⁸

WGS1363 showed resistance against ampicillin (MIC 16 mg/L), amoxicillin (MIC 32 mg/L) and piperacillin (MIC 128 mg/L) (cf. Table 1). Addition of β-lactamase inhibitors (clavulanate, sulbactam, tazobactam and avibactam) only reduced MICs by up to 0–4 dilution steps. MICs of cephalothin, cefuroxime, cefotaxime, ceftazidime, cefpirome, aztreonam, meropenem and mecillinam were all within the EUCAST wild-type distribution for E. coli.

The G239S substitution was previously obtained in a CTX-M-15 background in an error-prone PCR generated CTX-M-15 clone library with piperacillin/tazobactam selection.⁸ We used the original pZS3-CTX-M-15 and pZS3-CTX-M-178 plasmids from this study and additionally cloned bla_CTX-M-27 and bla_CTX-M-255 in the same plasmid backbone, but with the native CTX-M-27 promoter instead of the P3 promoter used by Rosenkilde et al. to obtain the pZS3*-CTX-M-27 and pZS3*-CTX-M-255. All four plasmids and the empty pZS3 control plasmid were expressed in E. coli TOP10 and MICs were determined (Table 1). E. coli TOP10/pZS3*-CTX-M-255 reproduced the susceptibility phenotype of the parental isolate WGS1363. Both pZS3-CTX-M-178- and pZS3*-CTX-M-255-transformants showed resistance to amoxicillin and piperacillin alone. Combination with β-lactamase inhibitors reduced MICs up to 0–4 dilution steps and appeared least active against CTX-M-255 in combination with piperacillin.

WGS1363 had a low β-lactamase activity (0.5 mU/mg) that was reduced only 11-fold following treatment with tazobactam (5 mg/L). In comparison EC101, a piperacillin/tazobactam-susceptible E. coli isolate containing a single copy of bla_TEM-1 with a P3 promoter,³ had a β-lactamase activity of 16 mU/mg that was reduced 107-fold by previous incubation with tazobactam. To further detail the enzymatic activity of CTX-M-variants with a G239S substitution, CTX-M-15, CTX-M-27, CTX-M-178 and CTX-M-255 were expressed with a C-terminal His-tag and affinity purified. Enzyme kinetic constants were determined for penicillin G, ampicillin, piperacillin, nitrocefin, cephalothin, cefotaxime and meropenem (Table 2). Both enzymes containing the G239S lacked measurable activity against cephalosporins or meropenem. In both enzymes, appreciable hydrolytic activity to piperacillin was conserved in contrast to hydrolytic activity to ampicillin that was reduced.

The IC₅₀ of clavulanate, sulbactam, tazobactam and avibactam was determined for using nitrocefin as a substrate. β-lactamase-inhibitors were less active against CTX-M-255 [IC₅₀s for clavulanate (55 µM), sulbactam (6644 µM), tazobactam (32 µM) and avibactam (119 µM)] compared to CTX-M-27 [IC₅₀s for clavulanate (0.10 µM), sulbactam (0.39 µM), tazobactam (0.024 µM) and avibactam (0.099 µM)]. A similar result was obtained for CTX-M-178 [IC₅₀s for clavulanate (108 µM), sulbactam (4430 µM), tazobactam (898 µM) and avibactam (195 µM)] compared to CTX-M-15 (IC₅₀s for clavulanate (0.009 µM), tazobactam (0.002 µM) and avibactam (0.059 µM); sulbactam inhibition assay was not done for this enzyme).

The phenotype of isolates containing bla_CTX-M-255 or similar enzymes showing resistance to inhibitors is subtle and may be attributed to the presence of OXA-enzymes, hyperproduction of TEM-enzymes or IRTs. We therefore queried genome databases unbiased to resistance phenotype. Sequence databases comprising 7313 Danish and 10 048 German clinical E. coli isolates that was whole-genome sequenced using Illumina short read sequencing was queried for G239S variants. The query identified three isolates, two containing blaCTX-M-178 (ST15331 SLV of ST131 and ST1193) and one containing the blaCTX-M-255 variant (ST131). Sequence data for the isolates are available at NCBI BioProject PRJNA1071552. The isolate carrying blaCTX-M-255 belonged to the ST131 clade A, was serotype O16:H5 and carried the fimH41 allele. The blaCTX-M-178-carrying isolates belonged to ST1193 and ST15331. ST15331 is a single locus variant of ST131 and the isolate was close related to ST131 clade A isolates. Along with these, it was serotype O16:H5 and contained the fimH41 allele. Similarly, a North American study of 147 E. coli isolates susceptible to cephalosporins identified a single isolate containing bla_CTX-M-255 in addition to an isolate containing bla_CTX-M-189 (with an S133G substitution) associated with inhibitor resistance.¹⁹ The G239S substitution has also been reported in the background of CTX-M-3 (CTX-M-187; NG056409) and CTX-M-55 (CTX-M-227; MN104597). The occurrence of a G239S substitution in different CTX-M backgrounds, and in diverse E. coli genetic lineages isolated in different countries, is indicative of biologically significant convergent evolution.

Acknowledgements

Carola E.H. Rosenkilde and Morten O.A Sommer, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark are gratefully acknowledged for providing the pZS3-CTX-M-15 and pZS3-CTX-M-178 plasmids.

Funding

This study was supported by the Novo Nordisk Foundation (NNF 18OC0033946 to S.H.).

Transparency declarations

None to declare.

Supplementary data

Supplementary Information is available as Supplementary data at JAC Online.

References

Author notes