In Vitro Activity of Cefepime-Enmetazobactam against Gram-Negative Isolates Collected from U.S. and European Hospitals during 2014–2015

Enmetazobactam, formerly AAI101, is a novel penicillanic acid sulfone extended-spectrum β-lactamase (ESBL) inhibitor. The combination of enmetazobactam with cefepime has entered clinical trials to assess safety and efficacy in patients with complicated urinary tract infections.

from 16 to 0.12 g/ml. The MIC 90 s for K. pneumoniae were reduced by at least eight doubling dilutions from Ͼ64 to 0.5 g/ml. E. cloacae and E. aerogenes MIC 90 s were reduced by four and by one doubling dilution, from 16 to 1 g/ml and from 0.5 to 0.25 g/ml, respectively. Enmetazobactam did not enhance the potency of cefepime against P. aeruginosa, the MIC 90 for both cefepime and cefepime-enmetazobactam being 16 g/ml. Enmetazobactam did not show intrinsic activity against Enterobacteriaceae or P. aeruginosa (data not shown).
The epidemiological cutoff (ECOFF) values for cefepime were determined for each species (18) and are reported in Table 2. Against E. coli and K. pneumoniae, the ECOFF values were 0.12 g/ml. The ECOFF values for E. aerogenes and E. cloacae were 0.12 and 0.25 g/ml, respectively, and 16 g/ml for P. aeruginosa.
Enmetazobactam restored the activity of cefepime against ESBL-producing isolates of E. coli and K. pneumoniae. For ESBL-producing isolates of E. coli, enmetazobactam lowered the cefepime MIC 90 by at least ten doubling dilutions from Ͼ64 to 0.12 g/ml and for ESBL-producing K. pneumoniae by at least seven doubling dilutions from Ͼ64 to 1 g/ml (Table 2). Applying the 2019 Clinical and Laboratory Standards Institute (CLSI) susceptible-dose dependent (SDD) breakpoint for cefepime of 8 g/ml, enmetazobactam shifted all but one ESBL-producing isolates from the resistant category to the susceptible category, thereby restoring the activity of cefepime toward these species. Cefepime-enmetazobactam had only limited activity against K. pneumoniae isolates containing genes encoding KPC (MIC 90 of Ͼ64 g/ml) and VIM (MICs of Ͼ64 g/ml) carbapenemases.
Enmetazobactam is more potent than tazobactam against ESBL-producing isolates of K. pneumoniae. The activities of enmetazobactam and tazobactam, both at fixed concentrations of 8 g/ml, were compared in combination with cefepime against the subset of ESBL-producing isolates of K. pneumoniae ( Fig. 1 and Table 2). Enmetazobactam shifted the MIC 90 of cefepime from Ͼ64 g/ml to 1 g/ml, whereas the shift for tazobactam was from Ͼ64 g/ml to 8 g/ml.
For the combined Enterobacteriaceae, Ͼ90% of isolates were susceptible to meropenem, ceftolozane-tazobactam, and ceftazidime-avibactam according to CLSI criteria. For cefepime, piperacillin-tazobactam, ceftazidime and gentamicin, the susceptibility of For E. coli, Ͼ90% of isolates were in the CLSI susceptible category for piperacillintazobactam, meropenem, ceftolozane-tazobactam, and ceftazidime-avibactam. Applying a breakpoint of 1 g/ml to cefepime-enmetazobactam inhibited 99.7% of all E. coli isolates. For K. pneumoniae, meropenem and ceftazidime-avibactam had Ͼ90% of isolates in the CLSI susceptible category. Applying breakpoints of 1 to 8 g/ml to cefepime-enmetazobactam resulted in cumulative inhibitions of 93.2 to 96.4%, respectively, for all K. pneumoniae isolates. At their CLSI breakpoints, Ͼ90% of E. aerogenes isolates were susceptible to cefepime, meropenem, ceftazidime-avibactam, gentamicin, and ciprofloxacin, whereas Ͼ90% of E. cloacae isolates were susceptible to meropenem, ceftazidime-avibactam, and gentamicin. Susceptibility of E. cloacae to ceftolozane-     Against the subset of E. coli with an ESBL genotype, only meropenem, ceftolozanetazobactam, and ceftazidime-avibactam had Ͼ90% of isolates in the CLSI susceptible category; for K. pneumoniae with an ESBL genotype, this was the case for meropenem and ceftazidime-avibactam only. Between 50 and 85% susceptible isolates were observed for piperacillin-tazobactam and gentamicin for E. coli, and for ceftolozanetazobactam for K. pneumoniae. The remaining comparators had less than 45% susceptible isolates by CLSI criteria for E. coli and K. pneumoniae with an ESBL genotype. Applying breakpoints of 1 to 8 g/ml for cefepime-enmetazobactam resulted in cumulative inhibitions of 99.1% for E. coli and 92.2 to 100% for K. pneumoniae with an ESBL genotype, respectively. The combination of cefepime with tazobactam resulted in cumulative inhibitions of 76.5 to 92.2%, respectively, for ESBL genotype K. pneumoniae.
Against the subset of K. pneumoniae isolates with a KPC genotype, only ceftazidimeavibactam had Ͼ90% of isolates in the susceptible category. For gentamicin 71.1% of these isolates were in the CLSI susceptible category and between 0 and 5% for the remaining comparators. Applying breakpoints of 1 to 8 g/ml for cefepimeenmetazobactam to K. pneumoniae isolates with a KPC genotype resulted in cumulative inhibitions of 6.7 to 42.2%, respectively.
For P. aeruginosa ceftolozane-tazobactam and ceftazidime-avibactam each had Ͼ90% of isolates in the CLSI susceptible category, and between 65 and 85% for the remaining comparators. Applying the cefepime breakpoint of 8 g/ml rendered 82.8% of isolates susceptible to cefepime-enmetazobactam.
Resistance to 3GCs leaves clinicians with limited empirical treatment options. Carbapenems are recommended for infections caused by ESBL-producing Enterobacteriaceae (21), which has contributed to the growing carbapenem consumption in high-income countries during the past 2 decades (9). The emergence and spread of carbapenem-resistant pathogens was predictable (8,22), and carbapenem-resistant infections have become a serious public health threat with ensuing morbidity and mortality (23,24). Sparing carbapenem usage is advised as part of antimicrobial stewardship programs (10). Piperacillin-tazobactam is a carbapenem-sparing option for infections caused by ESBL-producing E. coli and K. pneumoniae (25,26). However, the outcomes from the recent MERINO study do not support piperacillin-tazobactam as an alternative to meropenem in patients with bloodstream infections caused by ceftriaxoneresistant E. coli or K. pneumoniae (27).
The present study found that enmetazobactam restored the activity of cefepime, a 4th-generation cephalosporin, against recent United States and European clinical isolates of Enterobacteriaceae expressing diverse ESBLs. Applying the CLSI breakpoint for cefepime to cefepime-enmetazobactam revealed that this novel ␤-lactam/␤-lactamase inhibitor combination outperformed piperacillin-tazobactam and was as potent as meropenem toward the complete Enterobacteriaceae panel and toward the subset of ESBL-producing E. coli and K. pneumoniae isolates, though it showed limited activity against KPC-producing Enterobacteriaceae. The addition of enmetazobactam also enhanced substantially the in vitro efficacy of cefepime against E. cloacae, with a muchimproved MIC 90 compared to either piperacillin-tazobactam or ceftolozane-tazobactam and an MIC 90 comparable to that of ceftazidime-avibactam.
Conclusion. The results of this study suggest that cefepime-enmetazobactam may prove to be a valuable carbapenem-sparing option for empirical treatment of serious Gram-negative infections in settings with an elevated prevalence of ESBL-producing Enterobacteriaceae. The intrinsic activity of cefepime against AmpCs and OXA-48 (12,13) implies that cefepime-enmetazobactam also will be useful for treating infections caused by Enterobacteriaceae expressing these resistance mechanisms in conjunction with an ESBL. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to confirm the identity of the organisms (Bruker Daltonics, Bremen, Germany). MICs were determined by broth microdilution according to CLSI guidelines using frozen antimicrobial panels (28). The percentage of isolates susceptible to comparator antibiotics was determined according to 2019 CLSI and EUCAST breakpoints (19,20). Cefepime-enmetazobactam breakpoints have not yet been assigned. For purposes of comparison CLSI or EUCAST breakpoints for cefepime alone were applied to cefepimeenmetazobactam (see Results section). Quality control tests were performed with E. coli ATCC 25922, E. coli ATCC 35218, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853 each day of testing in compliance with CLSI guidelines (19). Cefepime-enmetazobactam MICs were determined using enmetazobactam at a fixed concentration of 8 g/ml; likewise, cefepime-tazobactam MICs were determined using tazobactam at a fixed concentration of 8 g/ml. Quality control ranges of cefepime-enmetazobactam have been approved by the CLSI for the aforementioned quality control strains (29). ECOFF values were determined as described previously (18) using the ECOFFinder_ XL_2010_v2.0 file (http://www.eucast.org/mic_distributions_and_ecoffs/) for Microsoft Excel v1812, reporting the ECOFF 99% rounded up to the next MIC.

MATERIALS AND METHODS
E. coli and K. pneumoniae isolates with a cefepime MIC of Ն1 g/ml were genotyped by multiplex PCR for genes encoding class A ESBLs (CTX-M, SHV, and TEM) and KPCs, MBLs (IMP, VIM, NDM, and SPM), AmpCs (ACC, CMY, DHA, FOX, and ACT), and class D (OXA-48-like ␤-lactamases), followed by sequencing using methods described previously (30). E. coli or K. pneumoniae isolates were classified as having an "ESBL genotype" if an isolate contained a gene encoding an ESBL according to the Bacterial Antimicrobial Resistance Reference Gene Database (31), irrespective of the presence of an AmpC and/or the OXA-48 gene sequence (32). Isolates were classified as having a "KPC genotype" if an isolate contained a gene encoding a KPC irrespective of the presence of an ESBL, AmpC and/or OXA-48 gene sequence.