Abstract
Background
Aztreonam/avibactam is one of the last therapeutic options for treating infections caused by NDM-like-producing Enterobacterales. However, PBP3-modified and NDM-producing Escherichia coli strains that co-produce CMY-42 have been shown to be resistant to this drug combination. The aim of our study was to assess the in vitro activity of cefepime/taniborbactam and cefepime/zidebactam against such aztreonam/avibactam-resistant E. coli strains.
Methods
MIC values of aztreonam, aztreonam/avibactam, cefepime, cefepime/taniborbactam, cefepime/zidebactam and zidebactam alone were determined for 28 clinical aztreonam/avibactam-resistant E. coli isolates. Those isolates produced either NDM-5 (n = 24), NDM-4 (n = 2) or NDM-1 (n = 2), and they all co-produced CMY-42 (n = 28). They all harboured a four amino acid insertion in PBP-3 (Tyr-Arg-Ile-Asn or Tyr-Arg-Ile-Lys).
Results
All strains were resistant to aztreonam/avibactam and cefepime, as expected. The resistance rate to cefepime/taniborbactam was 100%, with MIC50 and MIC90 being at 16 mg/L and 64 mg/L, respectively. Conversely, all strains were susceptible to cefepime/zidebactam, with both MIC50 and MIC90 at 0.25 mg/L. Notably, all strains showed low MICs for zidebactam alone, with MIC50 and MIC90 at 0.5 mg/L and 1 mg/L.
Conclusions
Our data highlighted the excellent in vitro performance of the newly developed β-lactam/β-lactamase inhibitor combination cefepime/zidebactam against aztreonam/avibactam-resistant E. coli strains, suggesting that this combination could be considered as an efficient therapeutic option in this context. Our study also highlights the cross-resistance between acquired resistance to aztreonam/avibactam and the cefepime/taniborbactam combination.
Introduction
Among the emerging antibiotic resistance mechanisms, the dissemination of enzymatic mechanisms such as MBLs in Enterobacterales is one of the main sources of concern.1 Among MBLs, the New Delhi MBL, NDM, is the most commonly reported among Escherichia coli isolates, and is often associated with nosocomial infections, leaving very few therapeutic options available.2 NDM enzymes confer resistance to all β-lactams including carbapenems, but do not hydrolyse aztreonam.3 However, clinical isolates producing MBLs very often co-produce other β-lactamases such as AmpC-type enzymes or ESBLs, compromising the use of aztreonam alone.4 The development of new inhibitors, such as avibactam, has enabled new β-lactam/β-lactam inhibitor (BL/BLI) combinations to be developed. Avibactam, which is a diazabicyclooctane (DBO) β-lactamase inhibitor, has the ability to inhibit many of the most common cephalosporinases and ESBLs, but not MBLs.5,6 Therefore, the commercial development of the new combination aztreonam/avibactam offers an interesting therapeutic option against MBL-producing Gram-negatives including NDM producers.7,8 Many in vitro studies identified this combination as one of the most effective against MBL-producing Enterobacterales. Hence, in a recent systematic review combining in vitro and in vivo studies, Mauri et al.8 reported a high antimicrobial activity of aztreonam/avibactam in 80% of MBL-producing Enterobacterales, particularly against NDM-like producers.
Nevertheless, recent studies identified NDM-like-producing E. coli isolates showing reduced susceptibility or resistance to aztreonam/avibactam.9,10 This non-susceptibility pattern is explained by modifications of the sequence of the PBP3 protein through specific amino acid insertions (Tyr-Arg-Ile-Asn or Tyr-Arg-Ile-Lys), in association with production of CMY-type AmpC-type β-lactamases (and particularly CMY-42) that possess significant hydrolytic activity against aztreonam.9 Considering the worldwide spread of such aztreonam/avibactam-resistant and NDM-like-producing E. coli, evaluation of other BL/BLI combinations has therefore to be considered.11
The recent development of novel BL/BLI combinations including cefepime/zidebactam (WCK 5107) and cefepime/taniborbactam (VNRX-5133) provides promising alternatives against MBL producers. Indeed, zidebactam belongs to the DBO family and possesses a dual effect of direct antibacterial activity and an ‘enhancer’ effect on the PBP2 while its β-lactam partner (i.e. cefepime or aztreonam) acts on PBP3. On the other hand, taniborbactam is a boronic acid derivative with an ability to inhibit MBLs, including NDM-like (except NDM-9) and VIM-like enzymes.12 Both BL/BLI combinations have been tested in several in vitro studies and are now undergoing Phase 2 or 3 clinical evaluations.13–15
In a recent study, Vázquez-Ucha et al. highlighted the effective activities of cefepime/zidebactam and cefepime/taniborbactam on 400 strains of carbapenemase-producing Enterobacterales. Cefepime/zidebactam and cefepime/taniborbactam displayed MICs ≤2 mg/L against 96.4% and 75%, respectively, of the tested MBL-producing clinical isolates.15
Because these very latest combinations of cefepime/zidebactam and cefepime/taniborbactam exhibited efficient activity against NDM-like producers (although acting by different mechanisms of action), and may soon be commercially available, the objective of our study was to assess the in vitro activity of those two combinations against aztreonam/avibactam-resistant E. coli strains resulting from PBP3-modified NDM-like and CMY-42 co-production.
Materials and methods
To achieve this objective, a total of 28 aztreonam/avibactam-resistant NDM-like-producing E. coli clinical isolates were included in this study. The strains originated from Angola (n = 9), Switzerland (n = 9), Pakistan (n = 6), France (n = 3) and Cameroon (n = 1). Those isolates produced either NDM-5 (n = 24), NDM-4 (n = 2) or NDM-1 (n = 2), and they all co-produced CMY-42 (n = 28). They all harboured a four amino acid insertion into the PBP3 protein after residue 333, either Tyr-Arg-Ile-Asn (n = 22) or Tyr-Arg-Ile-Lys (n = 6) or YRIK (n = 6), as previously published.9
MICs were determined by using broth microdilution for aztreonam, aztreonam/avibactam, cefepime, cefepime/zidebactam, cefepime/taniborbactam and zidebactam alone. Cefepime and aztreonam were purchased from Sigma-Aldrich (St Louis, MO, USA), and zidebactam (HY-120859) and taniborbactam (HY-109124) from MedChem Express (Luzern, Switzerland). The concentrations of the zidebactam, taniborbactam and avibactam β-lactamase inhibitors were all fixed at 4 mg/L.5,15 MICs were determined in triplicate using broth microdilution in Mueller–Hinton broth (Bio-Rad, Marnes-la-Coquette, France) for all antibiotics or antibiotic combinations listed above. Results were interpreted according to the latest EUCAST breakpoints, and susceptibility breakpoints for the novel BL/BLI combinations were defined by referring to the corresponding β-lactam breakpoints.16 MIC50 and MIC90 values were calculated for reporting results. The reference strain E. coli ATCC 25922 was used as quality control for all testing.
In addition to the E. coli clinical isolates, and in order to evaluate the impact of the different genetic features already proven to interfere with susceptibility to aztreonam/avibactam, the susceptibility to the novel BL/BLI combinations was determined using a series of E. coli MG1655 recombinant strains exhibiting either the Tyr-Arg-Ile-Asn or Tyr-Arg-Ile-Lys amino acid insertions into their PBP3 sequence.17 The blaCMY-42 gene was amplified by PCR using primers CMY-For (5′-AACACACTGATTGCGTCTGACG-3′) and CMY-Rev (5′-GGCAAAATGCGCATGGGATT-3′), and cloned into plasmid pTOPO (Invitrogen), and then the recombinant plasmids were electrotransformed into each of the three E. coli MG1655 backgrounds (WT, with Tyr-Arg-Ile-Asn insertion or with Tyr-Arg-Ile-Lys insertion).
Results and discussion
MIC values obtained for clinical isolates and recombinant E. coli strains are listed in Table 1. As expected, all clinical isolates were resistant to aztreonam, aztreonam/avibactam and cefepime. The respective MIC50 values were at 32, 8 and 256 mg/L, and MIC90 values at 64, 16 and 256 mg/L. Interestingly, all aztreonam/avibactam-resistant clinical isolates tested here showed cross-resistance to cefepime/taniborbactam, with MIC50 and MIC90 being at 16 mg/L and 64 mg/L, respectively. By contrast, cefepime/zidebactam and zidebactam alone remained very effective, with corresponding MICs still being very low (Table 1). The PBP3 modifications in those different recombinant strains did not impact the MICs for cefepime/zidebactam and zidebactam, highlighting the direct antibacterial activity of the latter, and confirming its enhancing effect on the E. coli PBP2. On the other hand, the four amino acid insertion into the PBP3 sequences significantly impacted the MICs of aztreonam, aztreonam/avibactam, cefepime and cefepime/taniborbactam, although not sufficiently to reach resistance breakpoints. Among the different clinical isolates tested, only those exhibiting combined resistance mechanisms including modification of PBP3 and co-production of NDM-like and CMY-42 enzymes were shown to be sufficient to confer resistance to cefepime/taniborbactam.
Table 1.
Susceptibility testing of clinical E. coli isolates and recombinant E. coli strains for the different BL/BLI combinations tested
| Strain | MIC (µg/mL)a | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| STb | Origin of isolationb | MBLc | Other β-lactamase(s)c | PBP3 insertion sequenced | ATM | AZA | FEP | FEP-ZID | FEP-TAN | ZID | |
| R27922 | — | — | — | — | — | ≤0.125 | ≤0.125 | ≤0.125 | ≤0.125 | ≤0.125 | 0.25 |
| MG1655 | — | — | — | — | — | ≤0.125 | ≤0.125 | ≤0.125 | ≤0.125 | ≤0.125 | 0.25 |
| MG1655 | — | — | — | — | YRIK | 1 | 1 | 0.5 | ≤0.125 | 0.5 | 0.25 |
| MG1655 | — | — | — | — | YRIN | 1 | 1 | 0.5 | ≤0.125 | 0.5 | 0.25 |
| MG1655 | — | — | — | CMY-42 | — | 32 | ≤0.125 | 0.5 | ≤0.125 | ≤0.125 | 0.25 |
| MG1655 | — | — | — | CMY-42 | YRIK | 32 | 2 | 2 | ≤0.125 | 0.5 | 0.25 |
| MG1655 | — | — | — | CMY-42 | YRIN | 32 | 2 | 2 | ≤0.125 | 0.5 | 0.25 |
| MG1655 | — | — | NDM-5 | — | — | ≤0.125 | ≤0.125 | 8 | ≤0.125 | 0.5 | 0.25 |
| MG1655 | — | — | NDM-5 | — | YRIK | 0.5 | 0.5 | 128 | ≤0.125 | 8 | 0.25 |
| MG1655 | — | — | NDM-5 | — | YRIN | 0.5 | 0.5 | 128 | ≤0.125 | 16 | 0.25 |
| R-461 | 167 | France | NDM-1 | CMY-42 | YRIN | 32 | 16 | 256 | ≤0.125 | 32 | 0.5 |
| R-3038 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 32 | 8 | 256 | ≤0.125 | 16 | 0.5 |
| R-3031 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 128 | 8 | 256 | ≤0.125 | 16 | 0.25 |
| N-185 | 361 | Switzerland | NDM-5 | CMY-42 | YRIN | 32 | 8 | 256 | ≤0.125 | 32 | 0.5 |
| N-590 | 167 | Switzerland | NDM-5 | CMY-42 | YRIN | 64 | 8 | 256 | ≤0.125 | 16 | 0.5 |
| R-460 | 648 | France | NDM-1 | CMY-42 | YRIN | >256 | 8 | >256 | ≤0.125 | 32 | 0.5 |
| R-3033 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 64 | 8 | 256 | ≤0.125 | 16 | 0.5 |
| R-3040 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 64 | 8 | 256 | ≤0.125 | 16 | 0.5 |
| R-3043 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 64 | 8 | 256 | ≤0.125 | 16 | 0.5 |
| R-3029 | 10 | Angola | NDM-5 | CMY-42, CTX-M-group 1 | YRIN | 32 | 8 | >256 | ≤0.125 | 16 | 0.5 |
| R-3039 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 16 | 8 | 256 | ≤0.125 | 16 | 0.5 |
| R-3048 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 16 | 8 | >256 | ≤0.125 | 16 | 0.5 |
| R-3054 | 10 | Angola | NDM-5 | CMY-42 | YRIN | 16 | 8 | >256 | ≤0.125 | 16 | 0.25 |
| N-57 | 405 | Switzerland | NDM-5 | CMY-42 | YRIK | 32 | 8 | >256 | ≤0.125 | 64 | 1 |
| R-466 | 405 | Cameroon | NDM-4 | CMY-42, CTX-M-15, OXA-1 | YRIK | >256 | 8 | >256 | ≤0.125 | 32 | 1 |
| R-2222 | 9747 | France | NDM-4 | CMY-42 | YRIK | >256 | 8 | >256 | ≤0.125 | 16 | 0.25 |
| 148C | 167 | Pakistan | NDM-5 | CMY-42, TEM-1B | YRIN | 32 | 8 | >256 | ≤0.125 | 64 | 0.25 |
| 272A | 167 | Pakistan | NDM-5 | CMY-42, TEM-1B | YRIN | 64 | 8 | >256 | ≤0.125 | 64 | 0.25 |
| 278A | 167 | Pakistan | NDM-5 | CMY-42, TEM-1B | YRIN | 32 | 8 | >256 | ≤0.125 | 32 | 0.25 |
| N1153 | 167 | Switzerland | NDM-5 | CMY-2, CTX-M-15, OXA-1, TEM-1B | YRIK | >256 | 8 | >256 | ≤0.125 | 64 | 0.125 |
| N1146 | 167 | Switzerland | NDM-5 | CMY-42, TEM-1B-like | YRIN | 32 | 8 | 128 | ≤0.125 | 16 | 0.125 |
| 240F | 205 | Pakistan | NDM-5 | CMY-42, TEM-1B | YRIK | 32 | 16 | 256 | ≤0.125 | 16 | 1 |
| N1013 | 361 | Switzerland | NDM-5 | CMY-42 | YRIN | 128 | 8 | 256 | ≤0.125 | 64 | 0.25 |
| N1416 | 405 | Switzerland | NDM-5 | CMY-42, CTX-M-15, OXA-1, TEM-1B | YRIK | >256 | 16 | >256 | ≤0.125 | 64 | 1 |
| 142A | 61 | Pakistan | NDM-5 | CMY-42, TEM-1B | YRIN | 64 | 8 | 256 | ≤0.125 | 32 | 0.5 |
| N1470 | 617 | Switzerland | NDM-5 | CMY-42 | YRIN | 64 | 16 | >256 | ≤0.125 | 16 | 0.25 |
| N1076 | 940 | Switzerland | NDM-5 | CMY-42, TEM-1B | YRIN | 64 | 8 | >256 | ≤0.125 | 64 | 1 |
| 246A | 2659 | Pakistan | NDM-5 | CMY-131, TEM-1B | YRIN | 64 | 8 | >256 | ≤0.125 | 8 | 0.125 |
Bold MIC values correspond to a significantly elevated MIC value in the recombinant E. coli strains compared with WT E. coli MG1655. ATM, aztreonam; AZA, aztreonam/avibactam; FEP, cefepime; FEP-TAN, cefepime/taniborbactam; FEP-ZID, cefepime/zidebactam; ZID, zidebactam. ZID and TAN were used at a fixed concentration of 4 µg/mL.
Dash indicates not applicable. ST, sequence type.
Dash indicates no β-lactamase.
Dash indicates no insertion of amino acids in the PBP3 sequence.
Conclusion
This study highlighted that the efficacy of the novel BL/BLI combination cefepime/taniborbactam, developed to be efficient against NDM-producing isolates, was significantly impacted by the same resistance mechanisms that have been shown to counteract the efficacy of aztreonam/avibactam, namely the co-production of CMY-42 and NDM and the modification of the PBP3 target. On the other hand, our data showed that cefepime/zidebactam was an excellent therapeutic option against those MBL producers, mainly related to the antibacterial and enhancing activity of zidebactam. This combination therefore constitutes one of the ultimate treatment options in such contexts.
Acknowledgements
We would like to thank Shionogi & Co., Ltd (Doshomachi 3-chome, Chuo-ku, Osaka 541-0045, Japan) for providing us with the isolates Escherichia coli MG1655 PBP3::YRIK and Escherichia coli MG1655 PBP3::YRIN.
Contributor Information
Christophe Le Terrier, Faculty of Science and Medicine, Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, University of Fribourg, Fribourg, Switzerland; Division of Intensive Care Unit, University Hospitals of Geneva, Geneva, Switzerland.
Patrice Nordmann, Faculty of Science and Medicine, Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, University of Fribourg, Fribourg, Switzerland; Swiss National Reference Center for Emerging Antibiotic Resistance, Microbiology Unit, Fribourg, Switzerland; Institute for Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland.
Mustafa Sadek, Faculty of Science and Medicine, Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, University of Fribourg, Fribourg, Switzerland; Department of Food Hygiene and Control, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.
Laurent Poirel, Faculty of Science and Medicine, Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, University of Fribourg, Fribourg, Switzerland; Swiss National Reference Center for Emerging Antibiotic Resistance, Microbiology Unit, Fribourg, Switzerland.
Funding
This work was financed by the University of Fribourg, Switzerland, and by the Swiss National Science Foundation (grant FNS 310030_1888801).
Transparency declarations
None to declare.
References
- 1. Nordmann P, Poirel L. Epidemiology and diagnostics of carbapenem resistance in gram-negative bacteria. Clin Infect Dis 2019; 69: S521–8. 10.1093/cid/ciz824 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Falcone M, Tiseo G, Antonelli Aet al. . Clinical features and outcomes of bloodstream infections caused by New Delhi metallo-β-lactamase-producing Enterobacterales during a regional outbreak. Open Forum Infect Dis 2020; 7: ofaa011. 10.1093/ofid/ofaa011 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Yong D, Toleman MA, Giske CGet al. . Characterization of a new metallo-ß-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009; 53: 5046–54. 10.1128/AAC.00774-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Dortet L, Poirel L, Nordmann P. Worldwide dissemination of the NDM-type carbapenemases in gram-negative bacteria. Biomed Res Int 2014; 2014: 249856. 10.1155/2014/249856 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Le Terrier C, Nordmann P, Poirel L. In vitro activity of aztreonam in combination with newly developed β-lactamase inhibitors against MDR Enterobacterales and Pseudomonas aeruginosa producing metallo-β-lactamases. J Antimicrob Chemother 2022; 78: 107–8. [DOI] [PubMed] [Google Scholar]
- 6. Yahav D, Giske CG, Gramatniece Aet al. . New β-lactam-β-lactamase inhibitor combinations. Clin Microbiol Rev 2021; 34: e00021. 10.1128/CMR.00021-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Wang X, Zhang F, Zhao Cet al. . In vitro activities of ceftazidime-avibactam and aztreonam-avibactam against 372 gram-negative bacilli collected in 2011 and 2012 from 11 teaching hospitals in China. Antimicrob Agents Chemother 2014; 58: 1774–8. 10.1128/AAC.02123-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Mauri C, Maraolo AE, Di Bella Set al. . The revival of aztreonam in combination with avibactam against metallo-β-lactamase-producing gram-negatives: a systematic review of in vitro studies and clinical cases. Antibiotics (Basel) 2021; 10: 1012. 10.3390/antibiotics10081012 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Sadek M, Juhas M, Poirel Let al. . Genetic features leading to reduced susceptibility to aztreonam-avibactam among metallo-β-lactamase-producing Escherichia coli isolates. Antimicrob Agents Chemother 2020; 64: e01659-20. 10.1128/AAC.01659-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Alm RA, Johnstone MR, Lahiri SD. Characterization of Escherichia coli NDM isolates with decreased susceptibility to aztreonam/avibactam: role of a novel insertion in PBP3. J Antimicrob Chemother 2015; 70: 1420–8. 10.1093/jac/dku568 [DOI] [PubMed] [Google Scholar]
- 11. Sadek M, Ruppé E, Habib Aet al. . International circulation of aztreonam/avibactam-resistant NDM-5-producing Escherichia coli isolates: successful epidemic clones. J Glob Antimicrob Resist 2021; 27: 326–8. 10.1016/j.jgar.2021.09.016 [DOI] [PubMed] [Google Scholar]
- 12. Le Terrier C, Gruenig V, Fournier Cet al. . NDM-9 resistance to taniborbactam. Lancet Infect Dis 2023; 10.1016/S1473-3099(23)00069-5 [DOI] [PubMed] [Google Scholar]
- 13. ClinicalTrials.gov . Study of cefepime-zidebactam (FEP-ZID) in complicated urinary tract infection (cUTI) or acute pyelonephritis (AP).2022. https://clinicaltrials.gov/ct2/show/NCT04979806
- 14. ClinicalTrials.gov . Safety and efficacy study of cefepime/VNRX-5133 in patients with complicated urinary tract infections (CERTAIN-1).2022. https://clinicaltrials.gov/ct2/show/NCT03840148
- 15. Vázquez-Ucha JC, Lasarte-Monterrubio C, Guijarro-Sánchez Pet al. . Assessment of activity and resistance mechanisms to cefepime in combination with the novel β-lactamase inhibitors zidebactam, taniborbactam, and enmetazobactam against a multicenter collection of carbapenemase-producing Enterobacterales. Antimicrob Agents Chemother 2022; 66: e0167621. 10.1128/AAC.01676-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. EUCAST . Breakpoint tables for determination of MICs and zone diameter v. 12.0. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf
- 17. Sato T, Ito A, Ishioka Yet al. . Escherichia coli strains possessing a four amino acid YRIN insertion in PBP3 identified as part of the SIDERO-WT-2014 surveillance study. JAC Antimicrob Resist 2020; 2: dlaa081. 10.1093/jacamr/dlaa081 [DOI] [PMC free article] [PubMed] [Google Scholar]