Impact of chromosomally encoded resistance mechanisms and transferable β-lactamases on the activity of cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations against Pseudomonas aeruginosa.
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Abstract
Objectives
We aimed to compare the stability of the newly developed β-lactams (cefiderocol) and β-lactam/β-lactamase inhibitor combinations (ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam) against the most clinically relevant mechanisms of mutational and transferable β-lactam resistance in Pseudomonas aeruginosa.Methods
We screened a collection of 61 P. aeruginosa PAO1 derivatives. Eighteen isolates displayed the most relevant mechanisms of mutational resistance to β-lactams. The other 43 constructs expressed transferable β-lactamases from genes cloned in pUCP-24. MICs were determined by reference broth microdilution.Results
Cefiderocol and imipenem/relebactam exhibited excellent in vitro activity against all of the mutational resistance mechanisms studied. Aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam proved to be more vulnerable to mutational events, especially to overexpression of efflux operons. The agents exhibiting the widest spectrum of activity against transferable β-lactamases were aztreonam/avibactam and cefepime/zidebactam, followed by cefepime/taniborbactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam. However, some MBLs, particularly NDM enzymes, may affect their activity. Combined production of certain enzymes (e.g. NDM-1) with increased MexAB-OprM-mediated efflux and OprD deficiency results in resistance to almost all agents tested, including last options such as aztreonam/avibactam and cefiderocol.Conclusions
Cefiderocol and new β-lactam/β-lactamase inhibitor combinations show promising and complementary in vitro activity against mutational and transferable P. aeruginosa β-lactam resistance. However, the combined effects of efflux pumps, OprD deficiency and efficient β-lactamases could still result in the loss of all therapeutic options. Resistance surveillance, judicious use of new agents and continued drug development efforts are encouraged.Free full text
Impact of chromosomally encoded resistance mechanisms and transferable β-lactamases on the activity of cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations against Pseudomonas aeruginosa
Abstract
Objectives
We aimed to compare the stability of the newly developed β-lactams (cefiderocol) and β-lactam/β-lactamase inhibitor combinations (ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam) against the most clinically relevant mechanisms of mutational and transferable β-lactam resistance in Pseudomonas aeruginosa.
Methods
We screened a collection of 61 P. aeruginosa PAO1 derivatives. Eighteen isolates displayed the most relevant mechanisms of mutational resistance to β-lactams. The other 43 constructs expressed transferable β-lactamases from genes cloned in pUCP-24. MICs were determined by reference broth microdilution.
Results
Cefiderocol and imipenem/relebactam exhibited excellent in vitro activity against all of the mutational resistance mechanisms studied. Aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam proved to be more vulnerable to mutational events, especially to overexpression of efflux operons. The agents exhibiting the widest spectrum of activity against transferable β-lactamases were aztreonam/avibactam and cefepime/zidebactam, followed by cefepime/taniborbactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam. However, some MBLs, particularly NDM enzymes, may affect their activity. Combined production of certain enzymes (e.g. NDM-1) with increased MexAB-OprM-mediated efflux and OprD deficiency results in resistance to almost all agents tested, including last options such as aztreonam/avibactam and cefiderocol.
Conclusions
Cefiderocol and new β-lactam/β-lactamase inhibitor combinations show promising and complementary in vitro activity against mutational and transferable P. aeruginosa β-lactam resistance. However, the combined effects of efflux pumps, OprD deficiency and efficient β-lactamases could still result in the loss of all therapeutic options. Resistance surveillance, judicious use of new agents and continued drug development efforts are encouraged.
Introduction
The main challenge regarding the treatment of severe Pseudomonas aeruginosa infections results from the remarkable ability of this pathogen to compromise the activity of all clinically available β-lactams through the selection of chromosomally encoded resistance mechanisms, the growing prevalence of strains producing ESBLs and carbapenemases and the emergence in hospitals worldwide of epidemic MDR/XDR P. aeruginosa clones (also known as high-risk clones).1,2 The recent introduction of ceftolozane/tazobactam and ceftazidime/avibactam in the clinical setting has alleviated the urgent need for agents to combat MDR/XDR P. aeruginosa strains. However, resistance to these new compounds has been increasingly reported, either due to the selection during treatment of mutations in the catalytic pocket of the intrinsic AmpC enzyme of P. aeruginosa (PDC) or due to the production of broad-spectrum β-lactamases from genes acquired by horizontal gene transfer.3–6
Newly approved compounds with antipseudomonal activity include cefiderocol, imipenem/relebactam, meropenem/vaborbactam and aztreonam/avibactam. Cefiderocol is a siderophore–cephalosporin conjugate with high antipseudomonal activity. This is owed to its increased internalization via iron uptake pathways (such as the PiuA/PiuC or PirA/PirR systems) and to its high stability against mutational resistance mechanisms and the production of broad-spectrum β-lactamases.7,8 Imipenem/relebactam and meropenem/vaborbactam are combinations of a carbapenem with, respectively, an innovative diazabicyclooctane- (DBO-) or a β-lactamase inhibitor derived from boronic acid- (BOR-). Both combinations have potent activity against Class A and C β-lactamases, particularly against key targets such as KPC producers.9 Aztreonam/avibactam is active against all types of β-lactamases due to the combination of an MBL-stable β-lactam partner with avibactam, which inactivates all serine-type enzymes that can hydrolyse aztreonam.10 New combinations based on a DBO- or BOR-type scaffold with antipseudomonal activity and an ultra-broad spectrum of therapeutic coverage, including MBL producers, are in the late stage of development: cefepime/taniborbactam11 and cefepime/zidebactam.12 Other inhibitors with promising activity are also under development: nacubactam (a DBO with intrinsic anti-PBP-2 activity)13 and xeruborbactam (a broad-spectrum BOR-type inhibitor).14 Representative figures of the chemical structures of all these β-lactams and β-lactamase inhibitors are shown in Figure S1 (available as Supplementary data at JAC Online).
Inclusion of these treatments in the available antibacterial arsenal may represent a step forward in combating P. aeruginosa infections. However, their specific activity against the main resistance mechanisms of P. aeruginosa has not yet been thoroughly investigated. We challenged these innovative treatments against a large collection of P. aeruginosa laboratory isolates expressing the most relevant mutational and transferable β-lactam resistance mechanisms and combinations of these to precisely identify their role in the treatment of this continuously evolving threat.
Methods
Collection of PAO1-derived mutants expressing single or multiple combinations of the most relevant chromosomally encoded β-lactam resistance mechanisms found in P. aeruginosa
We screened a collection of 17 previously constructed isogenic PAO1-derived knockout mutants expressing the main chromosomally encoded P. aeruginosa β-lactam resistance mechanisms. The collection included (i) single knockouts of the blaPDC gene and single, double or triple knockouts of its regulators (dacB, ampD, ampDh2, ampDh3, dacC and dacG), covering a wide range of blaPDC expression levels; (ii) single knockout mutants of the oprD porin gene or the negative local regulators (mexR, mexZ and nfxB) of the main resistance–nodulation–division efflux operons in P. aeruginosa (these latter showing overexpression of mexAB-oprM, mexXY or mexCD-oprJ, respectively); (iii) dual knockout mutants including the main combinations of these mechanisms. The characterization of hyperexpression and/or absence of the deleted genes was determined by RT–PCR or PCR plus Sanger sequencing in previous work.15–18 A P. aeruginosa transposon mutant of piuC from a previously described library19 was also included due to its previously associated role in the acquisition of cefiderocol resistance in P. aeruginosa.8
Construction of a collection of PAO1-derived isolates expressing the most relevant transferable broad-spectrum β-lactamases found in P. aeruginosa
To elucidate the specific impact of the acquired β-lactamases circulating in P. aeruginosa on the new agents, we designed a library of 27 isogenic PAO1 recombinant isolates expressing: the most relevant Ambler Class A (blaGES-1, blaGES-5, blaCTX-M-9, blaCTX-M-15, blaSHV-12, blaPER-1, blaVEB-1, blaKPC-2, blaKPC-3, blaKPC-31 and blaKPC-35), B (blaVIM-1, blaVIM-2, blaIMP-13, blaIMP-28, blaNDM-1 and blaNDM-5), C (blaCMY-2, blaFOX-4 and blaDHA-1) and D (blaOXA-2, blaOXA-10, blaOXA-14, blaOXA-15 and blaOXA-48) β-lactamase variants; and two blaKPC variants previously selected in vitro through the incubation of 2 KPC-producing Klebsiella pneumoniae clinical isolates for consecutive days in 10-mL Mueller–Hinton (MH) tubes containing incremental concentrations of imipenem/relebactam up to their 64×
baseline MIC (KPC-2 N132S and KPC-3 L167R).20 Although these variants have not yet been detected in the clinical setting, they have potential interest in the study of imipenem/relebactam resistance mechanisms. Each allele was amplified from a previously sequenced clinical isolate available in our laboratory. PCR products were digested and ligated to pUCP-24 (gentamicin resistance marker), a high-copy number shuttle vector that has been extensively used for the expression of β-lactamases and MIC comparisons in P. aeruginosa,21,22 and transformed into Escherichia coli TG1. Transformants were selected in LB agar containing 10-mg/L gentamicin. Recombinant plasmids were extracted, and insert sequences were checked by PCR and Sanger sequencing. Plasmids were electroporated into PAO1, and transformants were selected in LB agar containing 30-mg/L gentamicin. Plasmids were rechecked by PCR and restriction analysis. To obtain an overall view of the interplay between mutational and transferable resistance mechanisms, eight candidate plasmids carrying genes encoding the most widespread Class A, B and D enzymes in P. aeruginosa (blaGES-5, blaPER-1, blaKPC-3, blaVIM-1, blaIMP-28, blaNDM-1, blaOXA-14 and blaOXA-15) were also transferred in parallel to the PAO ΔdacB ΔoprD or PAO ΔmexR ΔoprD dual knockout mutants.
Antimicrobial susceptibility testing
MICs of ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam, aztreonam/avibactam, cefepime, cefepime/taniborbactam, cefepime/zidebactam, cefiderocol, imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam for all isolates were determined in triplicate by reference broth microdilution assays. MICs were determined using CAMHB, except for cefiderocol, for which a specifically prepared iron-depleted CAMHB was used.23 Tazobactam, avibactam, taniborbactam and relebactam were tested at a fixed concentration of 4 mg/L, whereas vaborbactam and xeruborbactam were tested at 8 mg/L. Zidebactam and nacubactam were tested at a 1:1 ratio with cefepime and meropenem, respectively. EUCAST v 14.0 clinical breakpoints and guidelines (http://www.eucast.org/clinical_breakpoints/) were used. To define susceptibility or resistance to combinations not yet approved, the P. aeruginosa EUCAST clinical breakpoints of their β-lactam partners were used. Reference strains E. coli ATCC 25922, E. coli NCTC 13353, K. pneumoniae ATCC BAA-2814 and P. aeruginosa ATCC 27853 were used as controls.
Statistical analysis
Statistical analysis of the antimicrobial activity of the compounds against the isolates studied was performed using Fisher's exact test with the GraphPad software (v 8.3.0).
Results
Impact of mutational resistance
A comparative assessment of the impact of the most relevant P. aeruginosa mutational β-lactam resistance mechanisms in the activity of the agents under study is summarized in Table Table1.1. The collection of mutants with chromosomal resistance mechanisms showed MICs at susceptibility levels to most of the tested agents. Meropenem and meropenem-based combinations were active against most of the mutants tested, with the exception of the isolate showing combined mexAB-oprM overexpression and OprD deficiency. Cefiderocol and imipenem/relebactam were the most potent agents, with the lowest MICs for the mutants studied. The mutant with an inactivated piuC gene showed a 16-fold increase in cefiderocol MIC compared with PAO1, with no effect on the MICs of the other β-lactams, evidencing the specific role of the PiuA/PiuC system in cefiderocol internalization. Aztreonam/avibactam, cefepime/taniborbactam and cefepime/zidebactam were very active against mutants showing blaPDC overexpression or OprD deficiency. However, their MICs were significantly affected by up-regulation of the mexAB-oprM, mexXY or mexCD-oprJ efflux operons. Overexpression of mexAB-oprM conferred borderline resistance to the three combinations, whereas the effects of mexXY and mexCD-oprJ overexpression were particularly evident for cefepime/taniborbactam. Vaborbactam, nacubactam and xeruborbactam did not improve the activity of meropenem against any of the mutants with an increased expression of blaPDC or efflux operons or an inactivated oprD gene. Statistical analysis revealed no significant differences for most of the compounds tested since the effect of the studied mutational mechanisms did not usually result in a change in the clinical categorization.
Table 1.
Antibiotic susceptibility data for PAO1-derived knockout mutants expressing the most relevant P. aeruginosa mutation-driven β-lactam resistance mechanisms
| MIC (mg/L)a | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Strain | Genotype | Phenotypeb | CAZ (R | C/A (R | C/T (R | ATM (R | A/A (R | FEP (R | F/T (R | F/Z (R | FDC (R | IMP (R | I/R (R | MEM (R | M/V (R | M/N (R | M/X (R |
| PAO1 | Wild-type | Wild-type | ≤1 | ≤1 | ≤0.5 | ≤4 | 2 | 2 | 2 | ≤1 | ≤0.125 | 1 | ≤0.125 | 0.5 | ≤0.125 | 0.25 | 0.25 |
| ΔampC | ampC knockout mutant | AmpC deficieny | ≤1 | ≤1 | ≤0.5 | ≤4 | 2 | 2 | 2 | ≤1 | ≤0.125 | 0.25 | ≤0.125 | 0.25 | 0.25 | 0.25 | 0.25 |
| ΔampD | ampD knockout mutant | ampC overexpression [↑ampC ≈ 50-fold] | 8 | 2 | 1 | ≤4 | 2 | 8 | 2 | 2 | ≤0.125 | 1 | 0.5 | 1 | 1 | 0.5 | 0.5 |
| ΔampD ΔampDh2 ΔampDh3 | ampD-ampDh2-ampDh3 knockout mutant | ampC overexpression [↑ampC ≈ 1000-fold] | 32 | 2 | 2 | 8 | ≤1 | 8 | 1 | 2 | ≤0.125 | 1 | 0.25 | 0.5 | 0.25 | 0.125 | 0.25 |
| ΔdacB | dacB (PBP4) knockout mutant | ampC overexpression [↑ampC ≈ 50-fold] | 16 | 2 | 1 | 8 | 4 | 16 | 2 | 4 | ≤0.125 | 1 | 0.5 | 1 | 1 | 0.5 | 0.5 |
| ΔdacB ΔampD | dacB (PBP4)-ampD knockout mutant | ampC overexpression [↑ampC ≈ 1500-fold] | 64 | 4 | 2 | 16 | 4 | 32 | 2 | 4 | ≤0.125 | 1 | 0.25 | 1 | 1 | 0.5 | 0.5 |
| ΔdacB ΔdacC | dual dacB-dacC (PBP4-PBP5) knockout mutant | ampC overexpression [↑ampC ≈ 500-fold] | 16 | ≤1 | 2 | 8 | ≤1 | 16 | 1 | 2 | ≤0.125 | 0.5 | 0.25 | ≤0.125 | ≤0.125 | 0.25 | ≤0.125 |
| ΔdacB ΔdacC ΔpbpG | triple dacB-dacC-dacG (PBP4-PBP5-PBP7) knockout mutant | ampC overexpression [↑ampC ≈ 1200-fold] | 16 | 2 | 2 | 16 | ≤1 | 8 | 1 | 2 | ≤0.125 | 0.5 | 0.25 | 0.25 | 0.25 | ≤0.125 | ≤0.125 |
| ΔmexR | mexR knockout mutant | mexAB-oprM overexpression [↑mexB ≈ 10-fold] | 8 | 4 | ≤0.5 | 16 | 8 | 8 | 8 | 8 | 0.25 | 1 | 0.25 | 2 | 1 | 2 | 2 |
| ΔampD ΔmexR | ampD-mexR knockout mutant |
ampC overexpression [↑ampC ≈ 50-fold] | 32 | 4 | 2 | 16 | 8 | 16 | 8 | 8 | ≤0.125 | 1 | 0.25 | 2 | 2 | 1 | 2 |
| ΔmexZ | mexZ knockout mutant | mexXY overexpression [↑mexY ≈ 15-fold] | 2 | ≤1 | 1 | ≤4 | 2 | 8 | 8 | 4 | ≤0.125 | 1 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| ΔnfxB | nfxB knockout mutant | mexCD-oprJ overexpression [↑mexD ≈ 150-fold] | ≤1 | 2 | 1 | ≤4 | 2 | 8 | 8 | 2 | ≤0.125 | 1 | 0.25 | 0.25 | ≤0.125 | 0.25 | 0.25 |
| ΔoprD | oprD knockout mutant | OprD deficiency | ≤1 | ≤1 | ≤0.5 | ≤4 | ≤1 | 2 | 2 | ≤1 | ≤0.125 | 8 | 0.25 | 4 | 2 | 2 | 2 |
| ΔampC ΔoprD | dual ampC-oprD knockout mutant | AmpC deficiency | ≤1 | ≤1 | ≤0.5 | ≤4 | ≤1 | 2 | 2 | ≤1 | ≤0.125 | 0.25 | ≤0.125 | 2 | 2 | 2 | 2 |
| ΔampD ΔoprD | dual ampD-oprD knockout mutant |
ampC overexpression [↑ampC ≈ 50-fold] | 16 | ≤1 | 1 | 8 | 2 | 4 | 2 | 2 | ≤0.125 | 16 | 1 | 4 | 2 | 2 | 4 |
| ΔdacB ΔoprD | dual dacB-oprD knockout mutant |
ampC overexpression [↑ampC ≈ 50-fold] | 32 | 2 | 2 | 8 | 2 | 16 | 2 | 4 | ≤0.125 | 8 | 1 | 4 | 4 | 4 | 4 |
| ΔmexR ΔoprD | dual mexR-oprD knockout mutant |
mexAB-oprM overexpression [↑mexB ≈ 10-fold] | 8 | 2 | 1 | 16 | 8 | 8 | 8 | 4 | 0.25 | 16 | 1 | 32 | 16 | 16 | 16 |
| ΔmexZ ΔoprD | dual mexZ-oprD knockout mutant |
mexXY overexpression [↑mexY ≈ 15-fold]+ | ≤1 | ≤1 | 1 | ≤4 | 2 | 8 | 8 | 4 | ≤0.125 | 16 | 1 | 4 | 4 | 2 | 2 |
| ΔpiuC | piuC transposon-mutant | Impaired iron uptake | ≤1 | ≤1 | ≤0.5 | ≤4 | 2 | 2 | 2 | ≤1 | 2 | 1 | ≤0.125 | 0.5 | ≤0.125 | 0.25 | 0.25 |
CAZ, ceftazidime; C/A, ceftazidime/avibactam; C/T, ceftolozane/tazobactam; ATM, aztreonam; A/A, aztreonam/avibactam; FEP, cefepime; F/T, cefepime/taniborbactam; F/Z, cefepime/zidebactam; FDC, cefiderocol; IMP, imipenem; I/R, imipenem/relebactam; MEM, meropenem; M/V, meropenem/vaborbactam; M/N, meropenem/nacubactam; M/X, meropenem/xeruborbactam.
aEUCAST v 14.0 breakpoints indicated.
bExpression levels were calculated relative to PAO1 in previous work.15–18
Impact of transferable β-lactamases
Comparative MIC data for the 27 PAO1 transformants expressing different β-lactamases are shown in Table Table2.2. Aztreonam/avibactam and cefepime/zidebactam demonstrated the broadest spectrum of activity, with all strains encoding a β-lactamase susceptible to both combinations. The addition of taniborbactam decreased the cefepime MICs to below the resistance breakpoint (P<
0.0001) for all strains except for those encoding SHV-12, VIM-1, IMP or NDM enzymes. Cefiderocol was active against transformants harbouring a wide array of β-lactamases, but borderline susceptibility or clinical resistance was noted for those producing SHV-12, PER-1, VEB-1, ceftazidime/avibactam-resistant KPC variants (KPC-31 and KPC-35), NDM enzymes and the extended-spectrum oxacillinases OXA-14 and OXA-15. Imipenem/relebactam was active against transformants carrying most Class A ESBLs, KPC carbapenemases and Class C and D enzymes with cephalosporinase activity but was not active against the KPC-2 N132S and KPC-3 L167R variants, MBLs or OXA-48. None of the meropenem-based combinations significantly potentiated the activity of the carbapenem alone (P
>
0.05). Meropenem/vaborbactam and meropenem/nacubactam were active against ESBLs and KPC enzymes, whereas xeruborbactam proved to be the most potent inhibitor against the transformants producing GES-5, KPCs or OXA-48. None of these inhibitors potentiated the activity of meropenem against MBL producers. Altogether, when comparing the susceptibility rates of cefiderocol and the new β-lactam/β-lactamase inhibitor combinations with that of ceftazidime/avibactam and ceftolozane/tazobactam, statistically significant higher activity was observed for aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, cefiderocol, meropenem/nacubactam and meropenem/xeruborbactam (P
<
0.05).
Table 2.
Antibiotic susceptibility data of PAO1-derived recombinant isolates expressing the transferable β-lactamases commonly encountered in P. aeruginosa
| MIC (mg/L)a | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Strain | Ambler Class | Phenotype | CAZ (R | C/A (R | C/T (R | ATM (R | A/A (R | FEP (R | F/T (R | F/Z (R | FDC (R | IMP (R | I/R (R | MEM (R | M/V (R | M/N (R | M/X (R |
| PAO1 | — | Wild-type | ≤1 | ≤1 | ≤0.5 | ≤4 | 2 | 2 | 2 | ≤1 | ≤0.125 | 1 | ≤0.125 | 0.5 | ≤0.125 | 0.25 | 0.25 |
| GES-1 | A | ESBL | 32 | 8 | 16 | ≤4 | 4 | 16 | 2 | 2 | 0.25 | 1 | 0.5 | 0.5 | 0.25 | 0.5 | 0.25 |
| GES-5 | A | Carbapenemase | 4 | ≤1 | 2 | ≤4 | 4 | 4 | 2 | 2 | ≤0.125 | 2 | 0.5 | 4 | 2 | 1 | 0.25 |
| CTX-M-9 | A | ESBL | 4 | ≤1 | 1 | 16 | 2 | 512 | 2 | 8 | ≤0.125 | 1 | 0.25 | 1 | 1 | 0.5 | 0.25 |
| CTX-M-15 | A | ESBL | 32 | ≤1 | 2 | 128 | 2 | 512 | 2 | 4 | 0.5 | 1 | 0.25 | 0.5 | 0.5 | 0.5 | 0.25 |
| SHV-12 | A | ESBL | >512 | 16 | 128 | >512 | 16 | >512 | 32 | 8 | 8 | 1 | 0.25 | 2 | 1 | 1 | 1 |
| PER-1 | A | ESBL | 512 | 32 | >256 | >512 | 16 | 128 | 2 | 4 | 2 | 1 | 0.5 | 0.5 | 0.5 | 0.5 | 0.25 |
| VEB-1 | A | ESBL | >512 | 32 | >256 | >512 | 8 | 256 | 4 | 4 | 2 | 1 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| KPC-2 | A | Carbapenemase | >512 | 4 | >256 | 512 | 4 | 512 | 2 | 8 | 0.5 | 64 | 1 | 64 | 4 | 2 | 0.25 |
| KPC-3 | A | Carbapenemase | >512 | 4 | >256 | >512 | 4 | 512 | 2 | 4 | 1 | 64 | 1 | 64 | 4 | 2 | 0.25 |
| KPC-31b | A | ESBL | >512 | 512 | >256 | 256 | 4 | 256 | 4 | 8 | 8 | 1 | 0.5 | 2 | 1 | 1 | 0.25 |
| KPC-35c | A | ESBL | >512 | 32 | >256 | 64 | 4 | 256 | 4 | 4 | 4 | 1 | 0.5 | 2 | 1 | 1 | 0.25 |
| KPC-2 N132S | A | Carbapenemase | 4 | ≤1 | 2 | 256 | 4 | 2 | 2 | 2 | ≤0.125 | >128 | 16 | 128 | 32 | 8 | 0.25 |
| KPC-3 L167R | A | Carbapenemase | >512 | 16 | >256 | 256 | 4 | 512 | 4 | 4 | 1 | >128 | 8 | 32 | 2 | 4 | 0.5 |
| VIM-1 | B | Carbapenemase | >512 | >512 | >256 | ≤4 | 4 | >512 | 64 | 4 | 2 | 16 | 8 | 32 | 32 | 32 | 32 |
| VIM-2 | B | Carbapenemase | 128 | 64 | >256 | ≤4 | 4 | 64 | 2 | 4 | 0.25 | 8 | 8 | 16 | 16 | 16 | 16 |
| IMP-13 | B | Carbapenemase | >512 | 512 | >256 | ≤4 | 4 | 256 | 256 | 8 | 0.5 | 8 | 8 | 16 | 16 | 16 | 16 |
| IMP-28 | B | Carbapenemase | >512 | >512 | >256 | ≤4 | 4 | >512 | 512 | 8 | 0.5 | 8 | 8 | 64 | 32 | 32 | 64 |
| NDM-1 | B | Carbapenemase | >512 | >512 | >256 | ≤4 | 4 | >512 | 32 | 8 | 8 | 32 | 32 | >128 | >128 | >32 | >128 |
| NDM-5 | B | Carbapenemase | >512 | >512 | >256 | ≤4 | 2 | >512 | 32 | 8 | 8 | 32 | 32 | >128 | >128 | >32 | >128 |
| CMY-2 | C | Extended-spectrum cephamycinase | 512 | 4 | 64 | 128 | 4 | 128 | 8 | 8 | 1 | 2 | 0.25 | 4 | 2 | 2 | 1 |
| DHA-1 | C | Extended-spectrum cephamycinase | 256 | ≤1 | 32 | 32 | 2 | 8 | 2 | 4 | ≤0.125 | 1 | 0.5 | 1 | 0.5 | 0.5 | 0.25 |
| FOX-4 | C | Extended-spectrum cephamycinase | 256 | 32 | 16 | 16 | 8 | 32 | 2 | 4 | 0.25 | 1 | 0.5 | 1 | 1 | 1 | 0.5 |
| OXA-2 | D | Narrow-spectrum oxacillinase | 64 | 4 | 2 | 64 | 2 | 8 | 2 | 2 | 1 | 4 | 1 | 8 | 8 | 4 | 2 |
| OXA-10 | D | Narrow-spectrum oxacillinase | 2 | ≤1 | 2 | 32 | 2 | 32 | 2 | 2 | 0.5 | 2 | 0.5 | 8 | 2 | 2 | 0.5 |
| OXA-14d | D | ESBL | 512 | 256 | 128 | 32 | 8 | 64 | 4 | 4 | 2 | 2 | 0.5 | 4 | 2 | 2 | 2 |
| OXA-15e | D | ESBL | 256 | 64 | 128 | 16 | 2 | 16 | 4 | 4 | 4 | 2 | 0.25 | 1 | 0.5 | 1 | 1 |
| OXA-48 | D | Carbapenemase | ≤1 | ≤1 | 1 | ≤4 | 4 | 8 | 2 | 4 | ≤0.125 | 16 | 8 | 64 | 32 | 16 | 0.5 |
CAZ, ceftazidime; C/A, ceftazidime/avibactam; C/T, ceftolozane/tazobactam; ATM, aztreonam; A/A, aztreonam/avibactam; FEP, cefepime; F/T, cefepime/taniborbactam; F/Z, cefepime/zidebactam; FDC, cefiderocol; IMP, imipenem; I/R, imipenem/relebactam; MEM, meropenem; M/V, meropenem/vaborbactam; M/N, meropenem/nacubactam; M/X, meropenem/xeruborbactam.
aEUCAST v 14.0 breakpoints indicated.
bKPC-31 is a D179Y variant of KPC-3.
cKPC-35 is a L169P variant of KPC-2.
dOXA-14 is a G157D variant of OXA-10.
eOXA-15 is a D149G variant of OXA-2.
Interplay between mutational and transferable β-lactam resistance
The MIC data for the eight dual PAO ΔdacB ΔoprD and eight dual PAO ΔmexR ΔoprD knockout mutants expressing the most widespread ESBLs and carbapenemases are summarized in Tables S1 and S2 (available as Supplementary data at JAC Online). The association between the expression of transferable β-lactamases and blaPDC or mexAB-oprM overexpression with oprD inactivation limited the activity of almost all these new compounds, as they were inactive against most of the transformants.
Discussion
Consistent with data from large-scale surveillance studies,24 our findings revealed that cefiderocol and imipenem/relebactam were the most stable agents against P. aeruginosa mutational mechanisms. In accordance with recent reports,8 inactivation of piuC conferred a 16-fold increase in the baseline cefiderocol MIC, providing evidence of the major role that the PiuA/PiuC system plays in cefiderocol internalization (but not in the other β-lactams). Our results also provided interesting data about the effect of efflux on new compounds. Probably the most concerning finding was the effect of the overexpression of the mexAB-oprM efflux operon since mutations inactivating mexR can be selected during therapy with different β-lactams and result in increased aztreonam/avibactam, cefepime/taniborbactam and cefepime/zidebactam MICs.25,26 Finally, our findings revealed that the novel meropenem-based combinations are not expected to play a prominent role against this pathogen, given that meropenem resistance in P. aeruginosa is mainly associated with carbapenemase-independent mechanisms.2
Regarding transferable β-lactam resistance, aztreonam/avibactam and cefepime/zidebactam outperformed the other combinations (none of the transformants were found to be resistant). Thus, although their activity may be limited to some extent by efflux systems, they are expected to expand the available options to overcome transferable β-lactam resistance in clinical P. aeruginosa strains. These considerations could also be extended to cefepime/taniborbactam, which was active against isolates with transferable resistance mechanisms but did not cover IMP-type enzymes. In this regard, recent findings demonstrated that cefepime/taniborbactam-resistant P. aeruginosa isolates usually produce IMP-type enzymes or accumulate multiple chromosomal mutations.26 In contrast to mutants with chromosomal resistance mechanisms, the effectiveness of cefiderocol was significantly affected by some of the transferable mechanisms analysed here. In accordance with previous findings,22 ESBLs, KPCs, NDMs and the Class D OXA-14 and OXA-15 enzymes had a significant impact on the cefiderocol MICs. Given that the genes encoding these enzymes are increasingly reported in P. aeruginosa strains worldwide,1 close monitoring of their spread is encouraged to extend the lifespan of cefiderocol.
Imipenem/relebactam was not active against transformants producing an MBL or OXA-48, as previously observed.24 This combination was also inactive against the transformants harbouring the variants recently described in vitro KPC-2 N132S and KPC-3 L167R, which have shown resistance to relebactam inhibition in terms of Kiapp and IC50.20 On the other hand, the poor performance of meropenem-based combinations against most of these transformants could be explained by the following: (i) the poor inhibitory potency of vaborbactam against most of the carbapenemases included in the collection (its activity is restricted to KPC enzymes)9; (ii) the low intrinsic activity of nacubactam against P. aeruginosa, owing to the higher intrinsic resistance of this pathogen (MICs of 32 mg/L when tested alone)13; (iii) the poor performance of xeruborbactam against most of the MBLs included (despite its previously observed efficacy in Enterobacterales), which has recently been associated to the activity of the MexAB-OprM efflux pump in P. aeruginosa.27 Finally, the interplay between mutational and transferable resistance resulted in challenging phenotypes against which almost none of the options evaluated here was active. Altogether, our findings reveal the complexity of developing new antipseudomonal β-lactam-based therapies and highlight the yet unresolved therapeutic gaps regarding this continuously evolving pathogen.
Acknowledgements
We gratefully acknowledge Merck Sharp & Dohme for providing us with relebactam powder. We are also grateful to Shionogi for providing us with cefiderocol powder.
Contributor Information
Lucía González-Pinto, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Isaac Alonso-García, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Tania Blanco-Martín, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Pablo Camacho-Zamora, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Pablo Arturo Fraile-Ribot, Servicio de Microbiología & Instituto de Investigación Sanitaria Illes Balears (IdISBa), Hospital Universitario Son Espases, Palma de Mallorca, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Michelle Outeda-García, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Cristina Lasarte-Monterrubio, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Paula Guijarro-Sánchez, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Romina Maceiras, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain.
Bartolome Moya, Servicio de Microbiología & Instituto de Investigación Sanitaria Illes Balears (IdISBa), Hospital Universitario Son Espases, Palma de Mallorca, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Carlos Juan, Servicio de Microbiología & Instituto de Investigación Sanitaria Illes Balears (IdISBa), Hospital Universitario Son Espases, Palma de Mallorca, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Juan Carlos Vázquez-Ucha, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Alejandro Beceiro, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Antonio Oliver, Servicio de Microbiología & Instituto de Investigación Sanitaria Illes Balears (IdISBa), Hospital Universitario Son Espases, Palma de Mallorca, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Germán Bou, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Jorge Arca-Suárez, Servicio de Microbiología & Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario A Coruña, A Coruña, Spain. CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Funding
This work was supported by the Instituto de Salud Carlos III (ISCIII, projects PI20/01212, PI21/00704, PI22/01212 and PI23/00851) and co-funded by the European Union. This work was also supported by Merck Sharp & Dohme (MSD) through the Investigator Initiated Studies Program. The research was also funded by Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC, CB21/13/00055 and CB21/13/00099), the Spanish Network of Research in Infectious Diseases (REIPI, N° RD16/0016/0004 and N° RD16/0016/0006), integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (ERDF) ‘A way of making Europe’. The study was also funded by Axencia Galega de Innovación (GAIN), Consellería de Innovación and Consellería de Economía, Emprego e Industria, Xunta de Galicia (IN607D 2021/12 to A.B. and IN607A 2016/22 to G.B.). L.G.-P. was financially supported by the ISCIII project PI21/00704 and the ISCIII PFIS program (FI23/00074). I.A.-G. was financially supported by the ISCIII Río Hortega program (CM21/00076) and the ISCIII Juan Rodés program (JR23/00036). T.B.-M. was financially supported by the ISCIII project PI20/00686 and the ISCIII Río Hortega program (CM23/00095). J.C.V.-U. was financially supported by GAIN-Xunta de Galicia (IN606B 2022/009). J.A.-S. was financially supported by the ISCIII Juan Rodés program (JR21/00026).
Transparency declarations
Merck Sharp & Dohme (MSD) provided relebactam powder and did not exercise any control over the conduct or reporting of the research. Shionogi provided cefiderocol powder and did not exercise any control over the conduct or reporting of the research. J.C.V.-U. has received honoraria for lectures and/or presentations from MSD. A.O. has received grants or contracts from MSD, Wockhardt and Shionogi and consulting fees and honoraria for lectures and/or presentations from MSD, Pfizer and Shionogi. G.B. has received funding and study materials from MSD, grants or contracts from MSD, Pfizer, ABAC Therapeutics and Roche, consulting fees and honoraria for lectures and/or presentations from MSD, Shionogi, Pfizer, Roche and Menarini, and support for attending meetings and/or travels from Pfizer. J.A.-S. has received honoraria for lectures and/or presentations from Shionogi and Advanz Pharma. All other authors: none to declare.
Supplementary data
Figure S1 and Table S1 and S2 are available as Supplementary data at JAC Online.
References
Articles from Journal of Antimicrobial Chemotherapy are provided here courtesy of Oxford University Press
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Funding
Funders who supported this work.
Axencia Galega de Innovación
Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (1)
Grant ID: CB21/13/00055
Consellería de Innovación, Consellería de Economía, Emprego e Industria (1)
Grant ID: IN607D2021/12
European Union
ISCIII (1)
Grant ID: PI21/00704
ISCIII-General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund
Investigator Initiated Studies Program
Juan Rodés program (1)
Grant ID: JR21/00026
Merck Sharp & Dohme
National Plan for Scientific Research, Development and Technological Innovation 2013-2016
Río Hortega program (1)
Grant ID: CM21/00076
Spanish Network of Research in Infectious Diseases
Xunta de Galicia (1)
Grant ID: IN606B-2022/009