Exploring the activity of a novel orally bioavailable β-lactam-β-lactamase inhibitor combination, ceftibuten-ledaborbactam, against Enterobacterales-carrying bla_OXA-48

ABSTRACT

Ledaborbactam is a novel boronic-acid inhibitor of class A, C, and D β-lactamases, including OXA-48, being developed in combination with ceftibuten. Herein, we examined the in vitro activity of this combination vs. other orally bioavailable compounds against 50 Enterobacterales-carrying bla_OXA-48. Ranked by MIC₉₀, ceftibuten-ledaborbactam was the most active (0.5 mg/L), followed by levofloxacin and tebipenem (8 mg/L), sulopenem (16 mg/L), and ceftibuten-clavulanic acid, ceftibuten and amoxicillin-clavulanic acid (≥32 mg/L).

IMPORTANCE

Carbapenemase-producing Enterobacterales (CPE) that produce class D OXA-48-like carbapenemases are among the most challenging pathogens to treat. Therefore, new compounds with in vitro activity against these bacteria are crucial due to the limited options available. In this study, we compared the in vitro antimicrobial activity of ceftibuten-ledaborbactam with that of other oral antibiotics, including tebipenem, sulopenem, ceftibuten-clavulanic acid, amoxicillin-clavulanic acid, and levofloxacin, against OXA-48-producing Enterobacterales. Our results show that ceftibuten-ledaborbactam has the most potent activity, with an MIC₉₀ of 0.5 mg/L.

OBSERVATION

β-Lactams are the largest and most clinically used class of antibiotics due to their tolerability, efficacy, and relatively low toxicity (1). β-Lactams inhibit penicillin-binding proteins (PBPs), disrupting cell-wall peptidoglycan metabolism and leading to bacterial cell death. The most common β-lactam resistance mechanism in Gram-negative bacteria is the production of β-lactamases that hydrolyze the amide bond of β-lactams, rendering the molecule incapable of binding the cellular target. Based on structural characteristics, β-lactamases are classified into four different classes. Class A, C, and D β-lactamases use serine as a nucleophile, while class B enzymes are Zn²⁺-dependent metallo-enzymes (2). To circumvent the effects of β-lactamases, β-lactamase inhibitors were developed and are given in combination with a partner β-lactam. Clinically available β-lactamase inhibitors possess one of three major chemical scaffolds: β-lactam-based (e.g., tazobactam), boronic acid-based (e.g., vaborbactam), or diazabicyclooctane-based (e.g., avibactam) (2). Unfortunately, resistance to the clinically available β-lactam/β-lactamase inhibitor (BL/BI) combinations is emerging (3). Moreover, most are only available as intravenous formulations.

The combination of ceftibuten (a third-generation cephalosporin) and ledaborbactam etzadroxil (an orally bioavailable prodrug of a bicyclic boronate BLI) is in the clinical testing stages of development as an orally available BL/BLI for the treatment of complicated urinary tract infections (cUTI) (4). Ceftibuten was approved in 1995 by the Food and Drug Administration (FDA) for the treatment of chronic bronchitis, otitis media, pharyngitis, and tonsillitis (5). Ceftibuten has also been studied for the treatment of urinary tract infections (UTIs) alone or in combination with clavulanic acid (6–8). Ledaborbactam is a novel BLI with potent inhibitory activity against serine β-lactamases, as demonstrated by IC₅₀ values of <0.5 µM for all tested enzymes (9).

Ceftibuten combined with ledaborbactam displays in vitro antimicrobial activity against Enterobacteriaceae producing class A, C, and D β-lactamases, including extended-spectrum β-lactamases (ESBLs) and carbapenemases like KPC and OXA-48 (10–12). In murine infection models, the addition of ledaborbactam-etzadroxil to ceftibuten reduced bacterial burdens of ESBL- and KPC-2-producing E. coli and OXA-48 and KPC-producing Enterobacteriaceae (13). As carbapenemase-producing Enterobacterales (CPE) that produce class D OXA-48-like carbapenemases are one of the most problematic and difficult-to-treat β-lactamase-producing Gram-negative pathogens, compounds with in vitro activity against these pathogens are a welcome addition to the thinning drug arsenal. Therefore, in this study, we aimed to compare the in vitro antimicrobial activity of ceftibuten-ledaborbactam to that of other orally available antibiotics (tebipenem, sulopenem, ceftibuten-clavulanic acid, amoxicillin-clavulanic acid, and levofloxacin) against a challenge panel of OXA-48 CPE. The clinical isolates were purchased from Drs. Laurent Poirel and Patrice Nordmann at the University of Fribourg. The isolates were obtained between 2007 and 2012 from France, Lebanon, Morocco, Algeria, Switzerland, the Sultanate of Oman, Egypt, Libya, the Netherlands, and Turkey. Host sources include sputum, urine, rectal swab, pus, blood, placenta, and bronchoalveolar lavage fluid. This panel of 50 clinical isolates of Enterobacteriaceae (Escherichia coli, n = 13; Atlantibacter hermannii [formerly Escherichia hermannii], n = 1; Klebsiella oxytoca, n = 2; Klebsiella pneumoniae, n = 30; and Enterobacter cloacae, n = 4) was previously used to determine their susceptibility to cefepime-taniborbactam and other comparator antibiotics (14). In that previous work, we reported that these 50 clinical isolates carried bla_OXA-48, and 39/50 (78%) of them carried at least one ESBL gene (bla_SHV, bla_CTX-M, bla_OXA, bla_TEM, bla_VEB, and/or bla_CMY); bla_CTX-M-15 was by far the most common, being present in 32/50 (64%) of the isolates (14). Moreover, these 50 clinical isolates were also previously confirmed to produce active OXA-48 enzyme by MIC testing and immunoblotting with polyclonal anti-OXA-48 antibodies. Notably, the OXA-48 production level was heterogeneous among these strains (14). To build upon our previous work, we used a similar methodology to expand the susceptibility profile of these clinical OXA-48-producing strains to ceftibuten-ledaborbactam and other oral antibiotics.

Minimum inhibitor concentrations (MICs) for the bacterial isolates were determined by the Mueller-Hinton (MH) agar dilution method with overnight cultures grown in MH broth and diluted in MH broth to 10⁶ colony forming units (CFUs)/mL (15). The MIC measurements were performed using a Steers Replicator that delivered 10 μL of a diluted overnight culture containing 10⁴ CFUs per spot. Ledaborbactam was tested at a fixed concentration of 4 mg/L, while ceftibuten-clavulanic and amoxicillin-clavulanic acids were maintained at a fixed 2:1 ratio. K. pneumoniae ATCC 700603 carrying bla_SHV-18, K. pneumoniae ATCC BAA-1705 with bla_KPC, and E. coli ATCC 25922 were used as controls for BL/BI integrity, as recommended by the Clinical and Laboratory Standards Institute (CLSI). E. coli ATCC 25922 constitutively produces low levels of chromosomally encoded AmpC, a narrow-spectrum Ambler class C cephalosporinase (16). MIC data were interpreted using CLSI-established breakpoints when available. Accordingly, breakpoints for ceftibuten (susceptible [S] ≤ 8 mg/L; intermediate [I] = 16 mg/L; resistant [R] ≥ 32 mg/L) were used to assign phenotypes to the combinations with ledaborbactam and clavulanic acid. The breakpoints for the other combinations are as follows: amoxicillin-clavulanic acid (susceptible [S] ≤ 8/4 mg/L; intermediate [I] = 16/8 mg/L; resistant [R] ≥ 32/16 mg/L) and levofloxacin (susceptible [S] ≤ 0.5 mg/L; intermediate [I] = 1 mg/L; resistant [R] ≥ mg/L) (15). As breakpoints for tebipenem and sulopenem are not available, MIC data were not interpreted.

Results from the control strains confirmed the validity of our methods, as they all tested within the anticipated quality control ranges (Table S1). The MIC results from the 50 CPE isolates are shown in Table S2. Compared to all other agents tested, ceftibuten-ledaborbactam displayed the most potent activity against the testing panel of OXA-48 CPE isolates, with a MIC₉₀ value of 0.5 mg/L. As shown in Fig. 1, the next most active compounds based on MIC₉₀ were tebipenem and levofloxacin with a MIC₉₀ value of 8 mg/L, followed by sulopenem (MIC₉₀ 16 mg/L). Ceftibuten alone, ceftibuten-clavulanic acid, and amoxicillin-clavulanic acid were the least active compounds with an MIC₉₀ value ≥ 32 mg/L. Strikingly, the addition of ledaborbactam to ceftibuten decreased both the MIC₅₀ and MIC₉₀ at least 64-fold. When interpreted according to the CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints for ceftibuten, 98 and 96% of the strains, respectively, were considered as provisionally susceptible to the ceftibuten-ledaborbactam combination (Table 1). In contrast, all isolates tested resistant to amoxicillin-clavulanic acid, and 24% displayed resistance to ceftibuten alone; the addition of clavulanic acid to ceftibuten resulted in a 50% decrease in the number of resistant isolates.

Fig 1.

Line graph representing the cumulative % of isolates with a specific MIC vs. the tested antibiotics: CTB, ceftibuten; LED, ledaborbactam; TEB, tebipenem; SUL, sulopenem; CLA, clavulanic acid; AMC, amoxicillin-clavulanic acid; LVX, levofloxacin.

TABLE 1.

Agar dilution susceptibility testing results for 50 isolates of CRE-carrying bla_OXA-48^a

	CTB	CTB-LEDb	TEB	SUL	CTB-CLAc	AMCc	LVX
MIC90	>32	0.5	8	16	32/16	>32	8
MIC50	8	0.12	1	4	2/1	>32	8
% Susceptible (CLSI)	54	98	N/A	N/A	84	0	34
% Susceptible (EUCAST)	26	96	N/A	N/A	38	0	34
% Intermediate (CLSI)	22	0	N/A	N/A	4	0	6
% Resistant (CLSI)	24	2	N/A	N/A	12	100	60
% Resistant (EUCAST)	74	4	N/A	N/A	62	100	60

^a Breakpoints for CTB (CLSI: susceptible (S) ≤ 8 mg/L, intermediate (I) = 16 mg/L, resistant (R) ≥ 32 mg/L; EUCAST: S ≤ 1 mg/L, R > 1 mg/L) were used to assign phenotypes to the combinations with ledaborbactam and clavulanic acid. The breakpoints for the other combinations are as follows: AMC (CLSI: S ≤ 8/4 mg/L, intermediate (I) = 16/8 mg/L, R ≥ 32/16 mg/L; EUCAST: S ≤ 1 mg/L, R >1 mg/L), and LVX (CLSI: S ≤ 0.5 mg/L, I = 1 mg/L, R ≥ 2 mg/L; EUCAST: S ≤ 0.5 mg/L, R > 1 mg/L). N/A, not applicable.

^b Ledaborbactam was tested at a fixed concentration of 4 mg/L.

^c CTB-CLA and AMC were maintained at a fixed 2:1 ratio. CTB, ceftibuten; LED, ledaborbactam; TEB, tebipenem; SUL, sulopenem; CLA, clavulanic acid; AMC, amoxicillin-clavulanic acid; and LVX, levofloxacin.

Notably, compared to our previous published data obtained with these same clinical isolates using the same methodology with other non-orally available compounds (14), ceftibuten-ledaborbactam was also the most active combination in vitro against this panel of isolates. Indeed, ceftibuten-ledaborbactam is more active than ceftazidime-avibactam (MIC₉₀ 1 mg/L), cefepime-taniborbactam (MIC₉₀ 4 mg/L), and meropenem-vaborbactam (MIC₉₀ 16 mg/L). This comparison is shown in Fig. 2. Interestingly, the only strain that was provisionally resistant to ceftibuten-ledaborbactam, E. coli 165, tested susceptible to ceftazidime-avibactam (14). However, ceftazidime-avibactam-resistant E. coli MLI and K. pneumoniae ELS were provisionally susceptible to ceftibuten-ledaborbactam. Differential expression of bla_OXA-48, combined with other β-lactam resistance mechanisms, might explain these seemingly contradictory resistance profiles.

Fig 2.

Line graph representing the cumulative % of isolates with a specific MIC vs. previously tested non-orally available antibiotics: CTB, ceftibuten; LED, ledaborbactam; FEP, cefepime; FEP-TAN, cefepime-taniborbactam; MVB, meropenem-vaborbactam; CZA, ceftazidime-avibactam.

Our study has two relevant limitations. First, we used a collection of clinical strains that, although geographically and genetically diverse, were collected more than 10 years ago. As aforementioned, we opted to work with these isolates because we had previously demonstrated that they produced active OXA-48, and we had tested other antibiotics using the same method. Thus, the two data sets are comparable. Second, although the β-lactamase content of each isolate is known, other critical mechanisms of β-lactam resistance, such as porin alterations, are not. The lack of this information prevents us from fully explaining unexpected phenotypes (e.g., strains from the same species and identical β-lactamase content that display resistance/susceptibility to the same agent).

In conclusion, our results corroborate earlier reports on the high efficacy of ceftibuten-laborbactam against CPE, specifically against strains producing OXA-48. Furthermore, our study compares the in vitro activity of this novel combination with other oral and non-oral antibiotics. We found that ceftibuten-laborbactam is the most potent treatment for our challenging clinical isolates, as evidenced by the lowest MIC₅₀ and MIC₉₀ values. Given the current lack of an orally administered β-lactam or a β-lactam-β-lactamase inhibitor combination effective against CPE, our findings are promising. This combination could significantly enhance oral step-down therapy or prevent hospitalization altogether, ultimately improving the quality of life for patients infected with these difficult-to-treat pathogens.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.03148-25.

Supplemental material. spectrum.03148-25-s0001.docx.

Tables S1 and S2.

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