Efficacy of cefiderocol in combination with xeruborbactam versus taniborbactam against cefiderocol-resistant NDM-producing Pseudomonas aeruginosa

TL;DR

This study compares two drugs in restoring effectiveness of an antibiotic against a drug-resistant strain of Pseudomonas aeruginosa.

Contribution

The study reveals that taniborbactam, but not xeruborbactam, effectively reverses resistance in NDM-producing Pseudomonas aeruginosa.

Findings

Taniborbactam restored cefiderocol susceptibility in 95.2% of NDM-positive Pseudomonas aeruginosa isolates.

Xeruborbactam had no effect on reversing resistance in these isolates.

An isolate unresponsive to taniborbactam carried multiple copies of blaNDM-1.

Abstract

We evaluated the in vitro activity of cefiderocol in combination with either taniborbactam or xeruborbactam against cefiderocol-resistant, blaNDM-positive Pseudomonas aeruginosa clinical isolates from Vietnam and Nigeria. Taniborbactam restored cefiderocol susceptibility in most isolates (20/21, 95.2%), while xeruborbactam had no effect. Resistance was reversed by dipicolinic acid, which confirmed New-Delhi Metallo-β-Lactamase (NDM-mediated) resistance. One isolate that was unresponsive to taniborbactam carried multiple copies of blaNDM-1. These findings highlight the species-specific limitations of xeruborbactam in P. aeruginosa.

Tables

TABLE 1. Susceptibility testing results of non-Pseudomonas quality control strainsa

Non-Pseudomonas control strains	CFD	CFD + XER	CFD + TAN	CFD + DPA	CEP	CEP + TAN	MEM	MEM + XER	MLST
E. coli NCTC13353	0.25	–b	<=0.03	–	>=64	0.5	–	–	–
K. pneumoniae BAA-2814	1	0.125	–	–	–	–	>=64	0.06	–
K. pneumoniae Klpn003	8	2	2	1	–	–	–	–	101
E. cloacae etcl_1	4	1	0.5	0.25	–	–	–	–	96

TABLE 2. Susceptibility testing of cefiderocol and its combinations with either xeruborbactam (XER), taniborbactam (TAN), or DPA, for the studied NDM-positive Pseudomonas aeruginosa isolates and NDM-positive non-Pseudomonas quality control strains

P. aeruginosa isolates	MIC (mg/L)				MLST	Clustera
	Cefiderocol	Cefiderocol +XER	Cefiderocol +TAN	Cefiderocol +DPA
X273	32	32	16	0.5	233	C01
X351	0.5	0.5	0.06	<=0.03	1420	–b
X444	1	0.5	0.06	<=0.03	308	C04
X445	8	8	2	0.5	233	C01
X474	4	4	1	0.5	308	C03
X503	4	4	0.5	0.25	308	C03
X549	2	2	0.5	0.5	308	C04
X583	4	4	2	0.5	308	C03
X734	4	4	0.5	0.25	308	C03
X888	4	4	1	0.5	308	C03
X951	1	1	0.5	0.06	235	–
X2041	2	2	0.25	<=0.03	308	C03
X010_19	4	4	0.5	0.25	773	C02
X109_19	4	4	0.5	0.25	773	C02
X123_19	8	8	1	0.5	773	C02
X132_19	4	4	0.5	0.25	773	C02
X159_19	2	2	0.25	0.125	773	C02
X193_19	1	1	0.5	0.125	773	–
X201_19	8	8	1	0.125	773	C02
X202_19	16	16	2	0.5	773	C02
X204_19	8	8	1	0.25	773	C02
X205_19	4	4	2	1	773	C02
X230_19	4	4	0.5	0.06	773	C02
X235_19	2	2	0.5	0.25	773	–
X239_19	0.5	0.5	0.125	<=0.03	773	C02
X257_19	0.25	0.125	<=0.03	<=0.03	773	C02
X263_19	0.25	0.25	0.125	<=0.03	773	C02
X265_19	8	8	2	0.25	773	C02
X274_19	1	1	0.25	<=0.03	773	C02
X302_19	2	2	0.5	0.125	773	C02
X303_19	2	2	0.5	0.06	773	C02
X306_19	4	4	1	0.25	773	–
X309_19	4	4	0.5	0.5	773	C02
X312_19	1	1	0.5	0.125	773	C02
X314_19	1	1	0.25	0.06	773	C02
X324_19	2	2	1	<=0.03	773	–
X331_19	2	2	0.5	0.125	773	C02
X332_19	0.06	0.06	<=0.03	<=0.03	773	C02
X338_19	8	8	0.5	0.5	773	–
X341_19	1	1	0.5	<=0.03	773	–
X344_19	2	2	0.25	<=0.03	773	–
X352_19	2	2	0.5	0.06	773	C02
X359_19	4	4	0.5	0.5	773	C02
X361_19	1	1	0.25	<=0.03	773	C02
X362_19	0.5	0.5	0.125	<=0.03	773	C02
X363_19	0.5	0.5	0.125	<=0.03	773	C02

Full text

INTRODUCTION

Metallo-beta-lactamase (MBL)-producing gram-negative bacteria, particularly those expressing NDM, present a major clinical challenge due to the limited availability of alternative treatment options. One potential treatment option is cefiderocol, a siderophore-conjugated cephalosporin antibiotic with stability against hydrolysis by MBL. However, NDM production in Gram-negative bacteria is associated with elevated cefiderocol minimum inhibitory concentrations (MICs), and cefiderocol non-susceptibility is particularly high in NDM-producing Pseudomonas aeruginosa (1). In high-income countries, carbapenem resistance in P. aeruginosa is typically driven by mutations that lead to the overproduction of efflux pumps and the loss of porin rather than NDM production (2). In contrast, NDM- or other carbapenemase-producing P. aeruginosa is frequently encountered in many low- and middle-income countries (3). In low-income countries, the lack of alternatives to treat these extensively drug-resistant P. aeruginosa has led to the extensive use of colistin. However, the toxicity of colistin is a major issue that could be resolved by novel substances that can overcome the underlying resistance mechanisms, such as MBL production.

Currently, licensed novel β-lactamase inhibitors, e.g., avibactam, relebactam, and vaborbactam, lack activity against MBLs. However, two novel boronate-based β-lactamase inhibitors, taniborbactam and xeruborbactam, have shown promise in inhibiting MBLs, with the exception of NDM-9 and NDM-30 variants and IMP for taniborbactam and some IMP variants for xeruborbactam (4–6). While existing data on the in vitro activity of xeruborbactam in combination with β-lactams primarily focuses on the meropenem-xeruborbactam combination (7–9), phase 1 clinical trials evaluating xeruborbactam in combination with ceftibuten and cefiderocol are currently underway (NCT06079775 and NCT06547554, respectively). Preliminary results suggest that when combined with cefiderocol, xeruborbactam reduces the MIC of cefiderocol in Enterobacterales and Acinetobacter baumannii (10, 11). However, Le Terrier et al. showed that, when combined with cefepime, xeruborbactam was less effective than taniborbactam in reducing the MIC values of β-lactams in MBL-producing P. aeruginosa recombinant strains (12).

The efficacy of combining taniborbactam or xeruborbactam with cefiderocol against NDM-producing P. aeruginosa clinical isolates remains unexplored. This study investigates the in vitro activity of cefiderocol in combination with taniborbactam or xeruborbactam against NDM-producing, cefiderocol-resistant P. aeruginosa isolates obtained from clinical specimens in Vietnam and Nigeria.

34 NDM-producing P. aeruginosa isolates from Nigeria were obtained as part of a previous study (13). In Vietnam, 12 NDM-producing P. aeruginosa isolates were obtained from an unpublished collection of carbapenem-resistant isolates recovered between January and December 2021 from the intensive care units at the 108 Military Central Hospital in Hanoi. Detection of P. aeruginosa was performed in the routine microbiological diagnostic laboratory in Vietnam, using MALDI-TOF MS for identification and VITEK 2 for antimicrobial susceptibility testing. The isolates collected in Vietnam were sequenced using a hybrid methodology, and draft genomes and annotations were performed as previously published (see Supplementary Methods for more details) (14). To minimize sampling bias, only one isolate per patient was included from both study sites.

Cefiderocol susceptibility testing was performed on all isolates using the broth microdilution (BMD) method with iron-depleted cation-adjusted Mueller-Hinton broth (ID-CA-MHB), prepared in-house following a previously published protocol (15). For isolates resistant to cefiderocol, additional BMD testing was performed to evaluate the activity of cefiderocol in combination with a fixed concentration (4 µg/mL) of either xeruborbactam (Xeruborbactam disodium, MedChemExpress, USA) or taniborbactam (Taniborbactam hydrochloride, Biozol, Germany). To further assess the role of NDM in cefiderocol resistance, BMD was performed also using ID-CA-MHB supplemented with 100 mg/L dipicolinic acid (DPA), a known inhibitor of MBL activity. The EUCAST version 15.0 clinical breakpoint for cefiderocol against P. aeruginosa (>2 mg/L) was applied to interpret the results of both combinations. The reference strains P. aeruginosa ATCC27853 were used as a control for all susceptibility testing. Additionally, E. coli NCTC 13353, K. pneumoniae ATCC BAA-2814, and two previously characterized cefiderocol-resistant non-Pseudomonas isolates -blaNDM-5-positive Enterobacter cloacae (16) and blaNDM-1-positive Klebsiella pneumoniae- from our earlier studies and an unpublished cohort were included as external comparators to confirm the activity of xeruborbactam and taniborbactam in our BMD (Table 1).

The study included a total of 46 NDM-positive isolates. Of these, 21 isolates (45.7%) were resistant to cefiderocol (Table 2), with MIC values ranging from 4 to 32 mg/L. These results were consistent with previous studies reporting high cefiderocol resistance rates in NDM-positive P. aeruginosa (17). All isolates harbored the blaNDM-1 gene. Detailed genotypic resistance data for all isolates can be found in the Data S1.

The isolates belonged to five different sequence types (STs), with ST773 being the most prevalent overall. All of the isolates from Nigeria (34/34, 100%) belonged to ST773, while the most prevalent ST in Vietnam was ST308 (8/12, 66.6%) (Fig. S1). Cefiderocol-resistant isolates were identified in three different STs: ST773, ST308, and ST233. Average nucleotide identity analysis using a cut-off value of 99.99% revealed four genetic clusters, with cefiderocol-resistant isolates present in three different clusters, and two resistant isolates not belonging to any cluster. These findings suggest that cefiderocol resistance occurs in diverse genetic clones and is not limited to a single clonal lineage.

As NDM activity has previously been attributed to cefiderocol resistance (18), we investigated whether the inhibition of NDM by DPA could restore the cefiderocol susceptibility. Indeed, in the presence of DPA, the cefiderocol MICs for all resistant isolates were reduced to susceptible levels (Table 2), indicating that NDM was an important contributor to resistance.

We also investigated previously described cefiderocol resistance determinants beyond NDM (19). Comparative SNP analysis of cefiderocol-resistant versus susceptible isolates did not reveal unique mutations in most resistant isolates. However, some resistant isolates carried mutations previously associated with cefiderocol resistance (Data S3), suggesting that, while NDM was likely the primary resistance mechanism, other mechanisms may also contribute to the overall resistance phenotype. Two isolates (X273 and X445) carried the highest number of mutations and had elevated cefiderocol MICs (32 mg/L and 8 mg/L, respectively). However, in both cases, the MICs decreased to 0.5 mg/L in the presence of DPA. The full data set of mutations present only in the resistant isolates is available in the Data S3.

Next, we evaluated whether adding taniborbactam or xeruborbactam could restore cefiderocol activity. Except for one isolate (X273), which had the highest cefiderocol MIC in the cohort, the addition of taniborbactam substantially reduced cefiderocol MICs, shifting them into the susceptible range based on EUCAST breakpoints. In contrast, xeruborbactam had no impact on cefiderocol MICs in NDM-producing P. aeruginosa (Table 2). This species-specific lack of efficacy in P. aeruginosa aligns with findings by Le Terrier et al., who reported reduced xeruborbactam activity in recombinant P. aeruginosa strains, potentially due to active efflux via the wild-type MexAB–OprM multidrug efflux system (12). In our cohort, three types of frameshift mutations in mexR and nalD, the regulators of MexAB–OprM, were identified in 11 isolates with varying cefiderocol susceptibility (Data S2). However, xeruborbactam did not significantly change the cefiderocol MICs of these 11 isolates, indicating that these mutations have no impact on xeruborbactam activity.

We further investigated the potential underlying resistance mechanism in the isolate X273, in which the taniborbactam could not revert the cefiderocol MIC to susceptible levels. WGS analysis revealed multiple copies of the blaNDM-1 gene. Previous reports have linked high cefiderocol MICs to increased blaNDM gene copy number (18), suggesting that a similar mechanism could explain the persistent resistance despite the addition of taniborbactam. To support this hypothesis, we quantified blaNDM-1 mRNA expression in X273 and its genetically closest strain in the cohort, X445, as previously described (20). The expression level in X273 was 3- to 4-fold higher than in X445, suggesting that the higher NDM expression is responsible for the elevated cefiderocol MIC. Furthermore, testing with higher concentrations of taniborbactam reduced the cefiderocol MIC of X273 further: to 2 mg/L at 8 mg/L taniborbactam and to 1 mg/L at 16 mg/L taniborbactam.

One limitation of our study is the low genetic diversity of the isolates, which may restrict how widely the findings can be applied. However, we focused on blaNDM-positive P. aeruginosa, which restricts the global population. Our findings demonstrate that cefiderocol resistance in blaNDM-positive P. aeruginosa is primarily driven by NDM activity and cannot be overcome by combining it with xeruborbactam. Furthermore, a similar finding has recently been reported in IMP-producing P. aeruginosa (21). These highlight important differences in the efficacy of novel β-lactamase inhibitors. Evaluations of new antimicrobial combinations specific to pathogens are needed, especially in the context of NDM-mediated resistance.