# In vitro activity of cefepime-enmetazobactam on carbapenem-resistant Gram negatives

Rémy A Bonnin, Katy Jeannot, Anne Santerre Henriksen, Juan Quevedo, Laurent Dortet

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Rémy A Bonnin, Katy Jeannot, Anne Santerre Henriksen, Juan Quevedo, Laurent Dortet. In vitro activity of cefepime-enmetazobactam on carbapenem-resistant Gram negatives. Clinical Microbiology and Infection, 2025, 31 (2), pp.240-249. 10.1016/j.cmi.2024.09.031. hal-04935159

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Original article

*In vitro* activity of cefepime-enmetazobactam on
carbapenem-resistant Gram negatives

Rémy A. Bonnin <fnref n="1" />, <fnref n="2" />, <fnref n="3" />, Katy Jeannot <fnref n="4" />, <fnref n="5" />, Anne Santerre Henriksen <fnref n="6" />, Juan Quevedo <fnref n="7" />,
Laurent Dortet <fnref n="1" />, <fnref n="2" />, <fnref n="3" />, *

ARTICLE INFO

Article history:

Received 15 June 2024

Received in revised form

12 September 2024

Accepted 28 September 2024

Available online 5 October 2024

Editor: G. Lina

Keywords:

β-lactam

β-lactamase inhibitor

Acinetobacter baumannii

Carbapenemase

Ceftazidime-avibactam

Imipenem-relebactam

Meropenem-vorbactam

OXA-48

Pseudomonas

A B S T R A C T

**Objectives:** Cefepime-enmetazobactam is a new β-lactam/βlactamase inhibitor combination with broad-spectrum activity against multidrug-resistant Enterobacterales, including extended-spectrum β-lactamase producers. This study evaluated the *in vitro* activity of cefepime-enmetazobactam towards a collection of carbapenem-resistant Enterobacterales (CRE), *Pseudomonas aeruginosa* and *Acinetobacter baumannii* compared to the other β-lactam/β-lactamase inhibitor combinations.

**Methods:** The MIC of cefepime, cefepime-enmetazobactam, ceftazidime, ceftazidime-avibactam, meropenem, meropenem-vorbactam, imipenem, imipenem-relebactam, and ertapenem were determined by broth microdilution on 2212 CRE, including 2089 carbapenemase producers (1000 OXA-48-like, 49 KPC, 697 NDM, 180 VIM, 1 IMP, 9 IMI, and 158 multiple carbapenemases) and 123 CRE that do not produce carbapenemase received at the French National Reference Centre (from March 1, 2023 to August 31, 2023), 50 *P. aeruginosa*, and 30 *A. baumannii*. All strains were fully sequenced.

**Results:** We confirmed the absence of inhibitory activity of enmetazobactam towards metallo-β-lactamases. Cefepime-enmetazobactam and ceftazidime-avibactam exhibited a similar susceptibility (96.7% vs. 99.5%, respectively) on OXA-48-producers. Cefepime-enmetazobactam exhibited 66.9% and 63.3% susceptibility for CRE non-EPC and KPC, whereas those rates rose to 96.7%/95.9%, 93.4%/95.9%, and 95.9%/98.0% for ceftazidime-avibactam, imipenem-relebactam, and meropenem-vorbactam, respectively. Low MICs (≤0.25 mg/L) were obtained for ceftazidime-avibactam-resistant KPC variants. Cefepime-enmetazobactam did not display a significant added value when compared with cefepime alone on *Pseudomonas aeruginosa* and *Acinetobacter baumannii*.

**Discussion:** OXA-48 producers displayed high susceptibility to cefepime-enmetazobactam, which is similar to ceftazidime-avibactam, including for OXA-48 producers that coproduce a ceftazidime hydrolyzing enzyme (extended-spectrum β-lactamases or AmpC). *In vivo* experiments have to be implemented to confirm if cefepime-enmetazobactam might be a relevant alternative to ceftazidime-avibactam for the treatment of infections caused by OXA-48 producers. **Rémy A. Bonnin, Clin Microbiol Infect 2025;31:240**

© 2024 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Introduction

Antibiotic resistance is a critical issue in modern medicine [1]. The β-lactams are the most prescribed antimicrobials in both hospital and community settings [2,3] and carbapenems are considered the last-resort treatment for complicated infections

<footnote n="1">¹) Team "Resist" UMR1184 "Immunology of Viral, Auto-Immune, Hematological and Bacterial Diseases (IMVA-HB)", INSERM, Université Paris-Saclay, CEA, LabEx LERMIT, Le Kremlin-Bicêtre, France</footnote>

<footnote n="2">²) Bacteriology-Hygiene Unit, Bicêtre Hospital, Assistance Publique-Hôpitaux de Paris, Le Kremlin-Bicêtre, France</footnote>

<footnote n="3">³) Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-Producing Enterobacteriaceae, Le Kremlin-Bicêtre, France</footnote>

<footnote n="4">⁴) Bacteriology Unit, University Hospital of Besançon, Besançon, France</footnote>

<footnote n="5">⁵) Associated French National Reference Center for Antibiotic Resistance in Pseudomonas and Acinetobacter, Besançon, France</footnote>

<footnote n="6">⁶) Maxel Consulting ApS, Jyllinge, Denmark</footnote>

<footnote n="7">7) ADVANZ Pharma, France</footnote>

<footnote>* Corresponding author. Laurent Dortet, Bacteriology-Hygiene Unit, Bicêtre Hospital, Assistance Publique-Hôpitaux de Paris, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre CEDEX, France.</footnote>

<footnote>E-mail address: laurent.dortet@aphp.fr (L. Dortet).</footnote>
caused by Gram negatives. Carbapenem resistance can result from the production of extended-spectrum β-lactamases (ESBLs) or cephalosporinases, coupled with outer-membrane decreased permeability, or from the production of carbapenemases [4].

Similar mechanisms are observed for *Pseudomonas aeruginosa*, where overexpression of the intrinsic cephalosporinase combined with OprD porin defect, or carbapenemase production results in carbapenem resistance [5].

In *Acinetobacter baumannii*, carbapenem resistance predominantly involves carbapenemase production [6].

In Enterobacterales, the main carbapenemases include KPC enzymes, NDM, VIM, and IMP metallo-β-lactamases (MBL), and OXA-48-like enzymes [4]. In France, the most prevalent carbapenemases are OXA-48-like, NDM and VIM in Enterobacterales, and VIM, NDM, IMP, and GES in *P. aeruginosa* [7]. In *A. baumannii*, OXA-23, and NDM remain the most prevalent carbapenemases [7].

Recently, the emergence of carbapenemase producers triggered the discovery of novel β-lactamase inhibitors (BLI) [8], including diazabicyclooctane derivatives like avibactam and relebactam, boronic acid-derived BLI such as vaborbactam [8], and more recently enmetazobactam, a tazobactam derivative with enhanced bacterial cell penetration, increased potency, and extended inhibition spectrum against cephalosporinases [9–11]. Cefepime-enmetazobactam demonstrated superior clinical efficacy to piperacillin-tazobactam for the treatment of complicated urinary tract infection (cUTI) and pyelonephritis [12]. *In vitro*, cefepime-enmetazobactam has shown efficacy against Enterobacterales, such as ESBL producers and *P. aeruginosa*, [13]. Recently, cefepime-enmetazobactam has received marketing authorization in Europe for the treatment of adults with cUTIs, hospital-acquired pneumonia, including ventilator-associated pneumonia and bacteremia, and in the United States for the treatment of cUTI.

The aim of this study was to evaluate the *in vitro* activity of cefepime-enmetazobactam towards a large collection of carbapenem-resistant Enterobacterales (CRE), carbapenem-resistant *P. aeruginosa*, and carbapenem-resistant *Acinetobacter baumannii* and to compare it with other β-lactam/BLI combinations (ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, and meropenem-vaborbactam).

## Material and methods

### Strain collection

Our collection included 2212 nonduplicated consecutive Enterobacterales with decreased susceptibility to carbapenems (at least to one among imipenem, meropenem, and ertapenem) sent from any French laboratory for expertise to the French National Reference Centre a 6-month period (from March 1, 2023 to August 31, 2023) (Tables S1-S8). Carbapenemases were detected using the Carba NP test [14] and the NG-Carba5 test (NG-Biotech) [15]. Overexpression of cephalosporinases was confirmed using cloxacillin supplemented (250 mg/L) medium (bioMérieux) [16] and ESBL by observing synergy between ceftazidime or cefepime disks and disk containing clavulanate.

We also included a representative panel of 50 *P. aeruginosa* (Table S9) and 30 *A. baumannii* (Table S10) collected by French National Reference Centre and exhibiting diverse β-lactam resistance mechanisms.

### Sequencing and bioinformatics analysis

All strains were fully sequenced using Illumina's technology [17] and resistant determinants were determined using Resfinder v4.0 [18].

### Susceptibility testing and MIC determination

MICs were determined by broth microdilution for ceftazidime, ceftazidime-avibactam, cefepime, cefepime-enmetazobactam, ceftolozane-tazobactam, ertapenem, imipenem, imipenem-relebactam, meropenem, and meropenem-vaborbactam with a range from ≤0.25 to >16 mg/L, using a customized Sensititre microplate (Thermo Fisher Scientific). As recommended by EUCAST, BLI concentrations were fixed at 4 mg/L for tazobactam, avibactam, and relebactam and at 8 mg/L for vaborbactam and enmetazobactam. The results were interpreted according to the EUCAST guidelines as updated in 2024 [19]. For cefepime-enmetazobactam, the EUCAST breakpoints released in 2024 for Enterobacterales were used (susceptible for MIC ≤4 mg/L and resistant for MIC >4 mg/L).

## Results

### Description of the CRE collection

The collection of Enterobacterales (Table 1) included 123 CRE that do not produce carbapenemase (Table 1 and Table S1) and 2089 carbapenemase producers (CPE) (Table 1 and Tables S2-S8) that reflect the French epidemiology of CPE in 2023 (Table 1). The most prevalent carbapenemases were OXA-48-like enzymes, with OXA-48 remaining the main variant (n = 721), followed by OXA-181 (n = 133), and OXA-244 (n = 109), a variant with lower carbapenemase activity [20]. Although OXA-48 was distributed in many different species, OXA-244 was exclusively identified in *Escherichia coli* (Table 1 and Table S6) that do not correspond to a monoclonal expansion since the concomitant spread of several clones (ST-38, ST-69, and ST-131) have been reported (Table S6) [21]. Two families of Ambler class A carbapenemases were present in this collection: KPC (Table S2) and IMI (Table S7). As expected, the 49 KPC producers were mostly KPC-3-producing *Klebsiella pneumoniae* [22], and the 9 IMI producers were all ECC [23]. NDM producers were the second most prevalent CPE (n = 697) (Table 1 and Table S3). NDM-1 was widely distributed among Enterobacterales species, whereas NDM-5 is mostly produced by *E. coli* and NDM-7 by ECC. NDM-14 was exclusively identified in *K. pneumoniae* of ST-147 [24]. VIM enzymes (n = 180) included mostly VIM-1 (n = 88) and VIM-4 (n = 88) variants. Notably, *bla*<sub>VIM-4</sub> was mainly carried by ECC (71/88) (Table 1 and Table S4).

### *In vitro* activity of cefepime-enmetazobactam on OXA-48-like-producing Enterobacterales

In the 1000 OXA-48-producing Enterobacterales MIC >16mg/L were reported for 24.5% and 42.0% of strains for cefepime and ceftazidime, respectively (Fig. 1(a) and (b)). Combining enmetazobactam with cefepime led to a strong restoration of susceptibility, lowering the MIC<sub>90</sub> from >16 mg/L to 1 mg/L. Similar results were observed for the ceftazidime-avibactam combination (MIC<sub>90</sub> decreased from >16 mg/L to 1 mg/L) (Fig. 1(a) and (b)). As previously reported [25], imipenem-relebactam and meropenem-vaborbactam did not demonstrate any increased potency when compared with carbapenems alone (Fig. 1(c) and (d)).

Because OXA-48-like carbapenemases are not able to hydrolyze ceftazidime, cefepime, and aztreonam [26], OXA-48-producing Enterobacterales can be separated into the following: (i) OXA-48 producers with no acquired resistance to 3GC (Fig. S1(a)), (ii) OXA-48 producers that coproduce an ESBL (Fig. S1(b)), and (iii) OXA-48 producers that coproduce an acquired cephalosporinase (Fig. S1(c)). In the first group, high susceptibility to cefepime was observed with MIC<sub>50</sub> and MIC<sub>90</sub> at ≤0.25 mg/L and 2 mg/L,
<caption>**Table 1**</caption>

Resistance mechanisms associated to the 2212 nonduplicated consecutive Enterobacterales with decreased susceptibility to carbapenems.

<table><thead><tr><th rowspan="2">Bacterial species</th><th colspan="5">Non-CPE</th><th colspan="10">Carbapenemase producers</th></tr><tr><th>ESBL</th><th>ESBL + CASE</th><th>CASE</th><th>CASE ACQ</th><th>HYPER OXA-1</th><th>HYPER OXA-10</th><th>HYPER OXY</th><th>HYPER SHV-1</th><th>HYPER TEM-1</th><th>KPC-31</th><th>KPC-130</th><th>Total Non-CPE</th><th>KPC-2</th><th>KPC-3</th><th>NDM-1</th><th>NDM-4</th><th>NDM-5</th><th>NDM-7</th><th>NDM-9</th><th>NDM-14</th><th>NDM-19</th></tr></thead><tbody><tr><td>C. braakii</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C. farmeri</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C. freundii</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td><td>1</td><td>1</td><td>60</td><td>3</td><td>4</td><td>11</td><td></td><td></td><td></td><td></td></tr><tr><td>C. koseri</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C. youngae</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>E. cloacae complex</td><td>23</td><td>23</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>46</td><td>1</td><td></td><td>95</td><td>1</td><td>13</td><td>30</td><td></td><td></td><td></td><td></td></tr><tr><td>E. coli</td><td>4</td><td>2</td><td></td><td></td><td>8</td><td>1</td><td>2</td><td></td><td></td><td></td><td></td><td>20</td><td></td><td></td><td>47</td><td>1</td><td>209</td><td>7</td><td>2</td><td>1</td><td>2</td><td></td></tr><tr><td>K. aerogenes</td><td></td><td></td><td>8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>K. oxytoca</td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td><td></td><td>1</td><td>19</td><td></td><td>1</td><td>1</td><td></td><td></td><td></td><td></td></tr><tr><td>K. pneumoniae</td><td>23</td><td>1</td><td></td><td></td><td>6</td><td></td><td></td><td></td><td></td><td>11</td><td></td><td>41</td><td>3</td><td>41</td><td>93</td><td>2</td><td>32</td><td>16</td><td></td><td></td><td></td><td>35</td></tr><tr><td>K. variicola</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>M. morganii</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>R. planticola</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>S. marcescens</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>S. ureilytica</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P. rettgeri</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P. stuartii</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P. mirabilis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>L. adecarboxylata</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>K. cryocrescens</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>All</td><td>27</td><td>29</td><td>31</td><td>17</td><td>1</td><td>2</td><td>2</td><td>11</td><td>1</td><td>1</td><td>1</td><td>123</td><td>6</td><td>43</td><td>325</td><td>7</td><td>260</td><td>65</td><td>2</td><td>36</td><td>2</td></tr></tbody></table>

Non-CPE, Non-carbapenemase-producer Enterobacterales; ESBL, extended-spectrum β-lactamase; CASE, cephalosporinase; CASE ACQ, acquired cephalosporinase.

respectively (Fig. S1(a)). The cefepime MIC$_{90}$ was lower than those of ceftazidime (>16 mg/L), imipenem (2 mg/L), and meropenem (2 mg/L) (Fig. 1). The addition of enmetazobactam resulted in a moderate decrease in the MIC$_{90}$ (2 mg/L to 1 mg/L). At the opposite, the addition of avibactam led to a decrease in ceftazidime MIC$_{90}$ from >16 mg/L to 0.5 mg/L (Fig. S1). In the group of OXA-48-like producers that coproduce an ESBL (mostly CTX-M) (Table S6), enmetazobactam and avibactam addition led to a strong decrease of MIC$_{90}$ from >16 mg/L for cefepime and ceftazidime to 2 mg/L and 1 mg/L for cefepime-enmetazobactam and ceftazidime-avibactam, respectively (Fig. S1(b)). For OXA-48 producers that acquired a cephalosporinase, a significant difference can be observed in the ability of the BLI to restore the efficacy of its combined β-lactams (Fig. S1(c)). Avibactam strongly lowered the MIC$_{90}$ of ceftazidime from >16 mg/L to 1 mg/L. Because cefepime is not a good substrate for cephalosporinases, the MIC$_{90}$ of cefepime was only lowered from 8 mg/L to 2 mg/L through the addition of enmetazobactam.

### In vitro activity of cefepime-enmetazobactam on MBL-producing Enterobacterales

As observed for the other BL/BLI combinations, enmetazobactam addition has no impact on the MIC of its associated β-lactam (Figs. 2 and 3).

For NDM producers 86.2% and 82.1% of isolates had cefepime and cefepime-enmetazobactam MICs ≥16mg/L, respectively. Despite a slightly better susceptibility to cefepime was observed for NDM-1 producers when compared with NDM-5 producers (Fig. 2(b)), it did not result in a significant modification of the clinical categorization (>95% of NDM producers remained resistant to cefepime ± enmetazobactam).

VIM producers exhibited lower MICs for cefepime (and therefore cefepime-enmetazobactam) when compared with NDM producers (Fig. 3). As observed for NDM producers, no difference was found in terms of MIC distribution when enmetazobactam was combined to cefepime. Contrary to NDM, MIC distributions were different depending on the VIM variants. VIM-4 producers possessed lower MICs for cefepime when compared with VIM-1 producers (Fig. 3(b)). Accordingly, 18.2% and 68.0% of VIM-1 and VIM-4 producers possessed MICs under the EUCAST clinical breakpoint (≤4 mg/L).

### In vitro activity of cefepime-enmetazobactam on KPC-producing Enterobacterales

A total of 51 KPC producers were tested including 49 KPC variants with carbapenemase activity (6 KPC-2 and 43 KPC-3) (Table 1 and Table S2) and two ceftazidime-avibactam resistant variants of KPC deprived of carbapenemase activity (KPC-31 and KPC-130) (Table 1 and Table S1). A significant inhibition of KPC-2/-3 enzymes was observed for all BLI, such as avibactam, relebactam, and vaborbactam, but also enmetazobactam (Fig. 4). Of note, whereas more than 95% susceptibility was obtained for ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam, only 63.3% of KPC-2/-3-producing isolates possessed MIC ≤4 mg/L to cefepime-enmetazobactam.

Regarding the two ceftazidime-avibactam resistant variants of KPC, cefepime-enmetazobactam possessed MIC at ≤ 0.25 mg/L (Table S1).

### In vitro activity of cefepime-enmetazobactam on carbapenem-resistant non-carbapenemase-producing Enterobacterales

Excluding the two non-carbapenemase KPC variants-producing isolates, the *in vitro* activity of cefepime-enmetazobactam was tested on 121 CRE that do not produce carbapenemase (Table 1 and Table S1). Carbapenem resistance mechanisms are recapitulated in Table 1. Because all these resistance mechanisms involved β-lactamases that can be inhibited by avibactam, relebactam, vaborbactam, and enmetazobactam, all BL/BLI associations might be a relevant therapeutic option. According to EUCAST clinical breakpoints, the rate of susceptibility rose to 42.1% for cefepime-enmetazobactam, 91.6% for ceftazidime-avibactam, 5.8% for imipenem-relebactam, and 3.4% for meropenem-vaborbactam when compared with cefepime, ceftazidime, imipenem, and meropenem (Table 2). For each MIC, the gain of susceptibility attributable to the inhibitor ranged from 9.1% (MIC ≤0.25 mg/L) to 43.0% (MIC = 8 mg/L) for cefepime-enmetazobactam, from 10.7% (MIC ≤0.25 mg/L) to 90.9% (MIC = 8 mg/L) for ceftazidime-avibactam, from 3.3% (MIC = 16 mg/L) to 36.5% (MIC = 0.5 mg/L) for imipenem-relebactam, and from 3.3% (MIC = 16 mg/L) to 20.4% (MIC = 0.5 mg/L) for meropenem-vaborbactam. Considering the fold reduction of the MIC$_{90}$,
<table><thead><tr><th colspan="14">Carbapenemase producers</th><th colspan="2">All</th></tr><tr><th></th><th>VIM-1</th><th>VIM-4</th><th>VIM-12</th><th>VIM-19</th><th>IMP-22</th><th>OXA-48</th><th>OXA-162</th><th>OXA-181</th><th>OXA-204</th><th>OXA-232</th><th>OXA-244</th><th>OXA-484</th><th>OXA-1181</th><th>IMI-1</th><th>IMI-2</th><th>IMI-4</th><th>IMI-6</th><th>IMI-19</th><th>Multiple</th><th>Total CPE</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td><td>4</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td><td>4</td></tr><tr><td>1</td><td></td><td></td><td></td><td></td><td></td><td>199</td><td></td><td>41</td><td>10</td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>24</td><td></td><td>380</td><td>383</td></tr><tr><td>18</td><td>4</td><td></td><td></td><td></td><td>1</td><td>17</td><td></td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>22</td><td>22</td></tr><tr><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>45</td><td>71</td><td>2</td><td></td><td></td><td></td><td>82</td><td></td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td><td>2</td><td>1</td><td>2</td><td>1</td><td>5</td><td>357</td><td>403</td></tr><tr><td>4</td><td>3</td><td></td><td></td><td></td><td></td><td>172</td><td></td><td>53</td><td></td><td></td><td></td><td>109</td><td>18</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td>37</td><td>666</td><td>686</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>14</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>16</td><td>24</td></tr><tr><td>6</td><td></td><td></td><td></td><td></td><td></td><td>40</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>5</td><td>74</td><td>78</td></tr><tr><td>9</td><td>9</td><td></td><td></td><td></td><td></td><td>176</td><td>1</td><td>30</td><td></td><td>5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>85</td><td>537</td><td>578</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>8</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>11</td><td>12</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>5</td><td>5</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>1</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>6</td><td>6</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>2</td><td>2</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td><td>2</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>1</td></tr><tr><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>88</td><td>88</td><td>2</td><td>2</td><td>1</td><td>721</td><td>1</td><td>133</td><td>10</td><td>7</td><td>109</td><td>18</td><td></td><td></td><td>3</td><td>2</td><td>1</td><td>2</td><td>1</td><td>158</td><td>2089</td><td>2212</td></tr></tbody></table>

avibactam might be considered as the most effective inhibitor of ESBL and/or AmpC (10-fold) followed by enmetazobactam (6-fold), regebactam (6-fold), and vaborbactam (4-fold).

In vitro activity of cefepime-enmetazobactam on P. aeruginosa and A. baumannii

For both *P. aeruginosa* and *A. baumannii* isolates, the addition of enmetazobactam did not significantly restore the activity of cefepime (Tables S9 and S10).

Regarding the 50 tested *P. aeruginosa* carbapenem resistance mechanisms included the following: (i) production of ESBL such as SHV-2a, CTX-M, PER-1, BEL-1, VEB-1, and GES; (ii) KPC, VIM, NDM, and IMP carbapenemase (Table S9); and (iii) overexpression of the intrinsic cephalosporinase (PDC) and several combinations with overexpression efflux pumps (MexAB-OprM, MexXY-OprM), and OprD deficiency. A moderate effect (gain of two to three 2-fold dilution when compared with cefepime alone) was observed for only five out of 50 isolates (2 CTX-M-producers, two isolates overproducing their cephalosporinase and one SHV-2a producer).

The 30 carbapenem-resistant *A. baumannii* included carbapenemase producers of OXA (OXA-23, OXA-72, and OXA-58), GES (GES-7 and GES-11), and KPC type (KPC-2) and strains that overexpressed their intrinsic cephalosporinase ADC (Table S10). A significant MIC reduction (≥ two 2-fold dilution) related to enmetazobactam was observed only for four isolates, being the wild-type strain, an ADC-267 overproducer, an AdeABC efflux pump overproducer, and an OXA-201 (the intrinsic OXA-51-like enzyme) overproducer.

Discussion

Summary of the principal findings in light of what was published before

Although the clinical efficacy of cefepime-enmetazobactam demonstrated its relevance as therapy for the treatment of ESBL-producing Enterobacterales when compared with piperacillin-tazobactam [12], its role in the treatment of carbapenem-resistant Gram negatives was not reported yet. Here, we tested the efficacy of cefepime-enmetazobactam against a large collection of carbapenem-resistant Gram-negative isolates (2,212 CRE and CPE, 50 *P. aeruginosa* and 30 *A. baumannii*).

Our results confirmed the absence of inhibitory activity of enmetazobactam towards metallo-β-lactamases (mostly NDM and VIM) [8,10].

At the opposite, we demonstrated that cefepime-enmetazobactam might be considered as a relevant therapeutic option for the treatment of infections caused by OXA-48-producers. Indeed, since OXA-48-like carbapenemases are unable to hydrolyze ceftazidime, aztreonam or cefepime, a combination with a BLI able to strongly inhibit additional extended-spectrum β-lactamase might be highly relevant. On the 1000 consecutive OXA-48 producers from France, cefepime-enmetazobactam and ceftazidime-avibactam exhibited a similar activity.

Despite we confirmed that enmetazobactam was an inhibitor of KPC [11], cefepime-enmetazobactam was found to be less efficient than ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam towards KPC producers [11]. However, 31/49 KPC producers displayed susceptibility to cefepime-enmetazobactam (MIC ≤ 4mg/L). No correlation between cefepime-enmetazobactam susceptibility with the KPC type enzyme (KPC-2 or KPC-3) or with co-produced β-lactamases was found. Of note, regarding the two KPC isolates that produce ceftazidime-avibactam resistant KPC variants (KPC-31 and KPC-131) cefepime-enmetazobactam displayed very low MICs (≤0.25 mg/L) suggesting a potential interest of this combination in countries where such variants have already emerged (Italy and the United States) [27]. Despite most of these ceftazidime-avibactam resistant KPC variants displayed a concomitant abolition of carbapenemase activity [27,28], carbapenem susceptibility might still be impaired by outer-membrane decreased permeability that possesses less impact on cefepime uptake, resulting in ceftazidime-avibactam and carbapenem-resistant isolates remaining fully susceptible to cefepime-enmetazobactam [27].

For *P. aeruginosa* and *A. baumannii*, cefepime-enmetazobactam did not display a significant added value when compared with cefepime alone [11]. Enmetazobactam was able to significantly restore the susceptibility of cefepime of very few isolates, mostly those expressing ESBL that are mainly prevalent in Enterobacterales but still rare in nonfermenters (CTX-M and SHV-2). Indeed, no relevant inhibition activity was observed for the ESBL that are predominant in *Pseudomonas* and *Acinetobacter*, namely GES, VEB, and PER enzymes.
OXA-48-like carbapenemases (n=1000)

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** MICs and cumulative MIC distribution of OXA-48-like-producing Enterobacterales to cefepime/cefepime-enmetazobactam (a), ceftazidime/ceftazidime-avibactam (b), imipenem/imipenem-relebactam (c), and meropenem/meropenem-vaborbactam (d).</figcaption></figure>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** (a) MICs and cumulative MIC distribution of NDM-producing Enterobacterales to cefepime/cefepime-enmetazobactam. (b) MICs and cumulative MIC distribution of NDM-1 and NDM-5-producing Enterobacterales to cefepime/cefepime-enmetazobactam.</figcaption></figure>

Implications for practice or policy

EUCAST breakpoints have been recently published for cefepime-enmetazobactam [19]. Contrary to what was applied for ceftazidime-avibactam (breakpoint at 8 mg/L for ceftazidime-avibactam when compared with 4 mg/L for ceftazidime alone) the cefepime-enmetazobactam breakpoint (4 mg/L) was aligned to those of cefepime (S ≤ 1 mg/L, R > 4 mg/L) [29]. Relying on these conservative breakpoints will prevent the confusion observed for ceftazidime and ceftazidime-

avibactam with MBL producers (i.e. MIC at 8 mg/L for both ceftazidime and ceftazidime-avibactam leads to reporting resistance to ceftazidime and susceptibility to ceftazidime-avibactam despite no active role of the BLI). To avoid confusion, it seems rational to align breakpoint of BL/BLI association to their corresponding BL. Because this rational has been used for imipenem-relebactam, meropenem-vaborbactam, and now cefepime-enmetazobactam, it would be important to critically assess the clinical breakpoint of ceftazidime-avibactam and align it to ceftazidime alone.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** (a) MICs and cumulative MIC distribution of VIM-producing Enterobacterales to cefepime/cefepime-enmetazobactam. (b) MICs and cumulative MIC distribution of VIM-1 and VIM-4-producing Enterobacterales to cefepime/cefepime-enmetazobactam.</figcaption></figure>

Regarding VIM-producing Enterobacterales, we observed that 18.2% and 68.0% of VIM-1 and VIM-4 producers possess cefepime MIC under the EUCAST clinical breakpoint (≤4 mg/L). Accordingly, cefepime-enmetazobactam will be reported as susceptible. Thus, it would be relevant to add a warning indicating that cefepime-enmetazobactam (as the other BL/BLI association) should not be tested for MBL. Of note, since ceftazidime is a better substrate of VIM enzymes when compared with cefepime [30], all VIM producers possess ceftazidime MIC over the EUCAST clinical breakpoint (4 mg/L), resulting in reporting systematic resistance.

## Strengths and limitations

Despite our *in vitro* results, which highlighted that cefepime-enmetazobactam might be a relevant therapeutic option for the treatment of OXA-48-like-producing Enterobacterales, these *in vitro* results have to be confirmed by *in vivo* data. Notably, the efficacy of cefepime-enmetazobactam demonstrated recently in murine pneumonia induced by *K. pneumoniae* producing OXA-48 like enzymes is promising [31].
KPC carbapenemases (n=49)

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 4.** MICs and cumulative MIC distribution KPC-producing Enterobacterales to cefepime/cefepime-enmetazobactam (a), ceftazidime/ceftazidime-avibactam (b), imipenem/imipenem-relebactam (c), and meropenem/meropenem-vaborbactam (d).</figcaption></figure>
<table><caption>**Table 2**
Cumulative rate of susceptibility of carbapenem-resistant Enterobacterales that do not produce a carbapenemase.</caption><thead><tr><th rowspan="2">Antimicrobial</th><th colspan="8">MIC (mg/L)</th><th rowspan="2">MIC<sub>50</sub></th><th rowspan="2">MIC<sub>90</sub></th></tr><tr><th>≤0.25</th><th>0.5</th><th>1</th><th>2</th><th>4</th><th>8</th><th>16</th><th>>16</th></tr></thead><tbody><tr><td>Cefepime</td><td>0.8%</td><td>5.0%</td><td>6.6%</td><td>13.2%</td><td>24.8%</td><td>35.5%</td><td>50.4%</td><td>100.0%</td><td>16</td><td>>16</td></tr><tr><td>Cefepime-enmetazobactam</td><td>9.9%</td><td>21.5%</td><td>41.3%</td><td>53.7%</td><td>66.9%</td><td>78.5%</td><td>87.6%</td><td>100.0%</td><td>2</td><td>>16</td></tr><tr><td>Ceftazidime</td><td>0.0%</td><td>0.0%</td><td>0.8%</td><td>2.5%</td><td>5.0%</td><td>5.8%</td><td>11.6%</td><td>100.0%</td><td>>16</td><td>>16</td></tr><tr><td>Ceftazidime-avibactam</td><td>10.7%</td><td>22.3%</td><td>52.9%</td><td>76.9%</td><td>92.6%</td><td>96.7%</td><td>100.0%</td><td>100.0%</td><td>1</td><td>4</td></tr><tr><td>Imipenem</td><td>24.0%</td><td>41.3%</td><td>69.4%</td><td>76.9%</td><td>87.6%</td><td>93.4%</td><td>96.7%</td><td>100.0%</td><td>1</td><td>8</td></tr><tr><td>Imipenem-relebactam</td><td>54.9%</td><td>77.9%</td><td>89.3%</td><td>93.4%</td><td>96.7%</td><td>100.0%</td><td>100.0%</td><td>100.0%</td><td>≤0.25</td><td>2</td></tr><tr><td>Meropenem</td><td>28.3%</td><td>37.5%</td><td>49.2%</td><td>66.7%</td><td>80.8%</td><td>92.5%</td><td>95.8%</td><td>100.0%</td><td>2</td><td>8</td></tr><tr><td>Meropenem-relebactam</td><td>47.1%</td><td>57.9%</td><td>70.2%</td><td>86.0%</td><td>90.9%</td><td>95.9%</td><td>99.2%</td><td>100.0%</td><td>0.5</td><td>4</td></tr><tr><td>Ertapenem</td><td>2.5%</td><td>5.8%</td><td>22.3%</td><td>33.9%</td><td>40.5%</td><td>57.0%</td><td>71.9%</td><td>100.0%</td><td>8</td><td>>16</td></tr></tbody></table>

Colors correspond to clinical categorization according to EUCAST guideline: green for “Susceptible at standard dosage”; yellow for “Susceptible upon increased exposure” and red for “Resistant”

## Understanding possible mechanism and implications for future research

Cefepime-enmetazobactam treatment of OXA-48 producers-related infections might help to limit the usage of ceftazidime-avibactam which is now considered as the reference for the treatment of infection caused by OXA-48 producers [32,33]. The recently reported emergence of NDM-producing E. coli with elevated MIC towards aztreonam-avibactam [34] and cefiderocol [35] should alert on the necessity of saving the avibactam combination (aztreonam + ceftazidime-avibactam and aztreonam-avibactam in the future) for the treatment of infections caused by MBL producers. Indeed, the emergence and rapid spread of OXA-244-producing E. coli in Europe [21,36–48] will probably increase the ceftazidime-avibactam antimicrobial pressure, resulting in the future spread of the following: (i) NDM-producing E. coli with decreased susceptibility to aztreonam-avibactam and cefiderocol [35,49–51], and (ii) ceftazidime-avibactam resistant KPC-producing K. pneumoniae [27]. Perhaps, the use of cefepime-enmetazobactam as an alternative to ceftazidime-avibactam for the treatment of OXA-48 producers might help to limit this harmful trajectory.

## Author contributions

R.A.B., K.J., and L.D. participated in conceptualization, data analysis, data interpretation, and writing of the original draft. A.S.H. and J.Q. participated in review and editing. A.S.H., J.Q., and L.D. participated in funding acquisition.

## Transparency declaration

### Potential conflict of interest

A.S.H. is a consultant for ADVANZ Pharma. J.Q. is an employee of ADVANZ Pharma. LD. participated in the ADVANZ Pharma European Board in 2023. The other authors declare that they have no conflicts of interest.

### Financial report

This study was conceptualized and entirely performed by the French National Reference Centre which was partially sponsored by ADVANZ Pharma through an internal Investigator Research Grant.

## Acknowledgements

We would like to thank French National Reference Centre's technician for the technical assistance.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2024.09.031.

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