Abstract

Introduction

The global spread of Gram-negative bacteria with pan-drug resistance is a worrying concern [1]. In 2017, the World Health Organization (WHO) ranked carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannii in the critical global priority list of pathogens [2]. This matter of concern has promoted research and development in the area of antibiotic resistance including the development of novel antibiotics. Acquired resistance to carbapenems may be driven either by non-enzymatic mechanisms including loss of or weaker expression of porin-encoding genes, mutations in chromosomally encoded porin genes (such as OprD in P. aeruginosa, or OmpK35 and OmpK36 in Klebsiella pneumoniae), overexpression of genes encoding efflux pumps, or modifications of penicillin-binding proteins affecting the binding affinity of the β-lactams, particularly in A. baumannii [3–6]. However, the main concern regarding the mechanism of carbapenem resistance is the production of acquired carbapenemases, belonging to either Ambler class A (mainly KPC and GES), class B (i.e., NDM, VIM, IMP), or class D (i.e., OXA-23, OXA-24/40, OXA-48, and OXA-181) β-lactamases, particularly found in Enterobacterales or A. baumannii. Considering that those different resistance mechanisms are often combined, very few therapeutical options may sometimes be left, especially when dealing with infections caused by producers of metallo-β-lactamases (class B).

The new siderophore cephalosporin cefiderocol is one of the few antibiotics being considered for treating infections caused by these so-called difficult-to-treat (DTR) Gram-negative pathogens, since this molecule is not significatively hydrolyzed by most β-lactamases, including carbapenemases. In addition, cefiderocol bactericidal activity can overcome most non-enzymatic resistance mechanisms (porin mutations, and efflux pumps) [7]. Nevertheless, several mechanisms of resistance to cefiderocol have been recently reported, such as porin mutations, mutations affecting siderophore receptors, efflux pump overproduction, and modifications of the target (penicillin binding protein 3, or PBP-3), or production of some given β-lactamases [8]. A wide range of β-lactamases including class A (PER, BEL, SHV, KPC), class B (NDM, VIM), class C (AmpC), or class D β-lactamases (OXA-2, OXA-10, OXA-46) has been demonstrated to contribute to reduce the susceptibility to cefiderocol [8–11]. In addition, the progressive in vivo development of reduced susceptibility to cefiderocol has been reported in clinical contexts, particularly within two clinical trials [12, 13].

Recently, many promising β-lactamase inhibitors have been developed and might be soon clinically available along with respective β-lactam partner molecules, providing novel clinically relevant β-lactamase/β-lactamase inhibitor (BL/BLI) combos, useful to treat infections caused by extensively drug-resistant strains [14]. Among the newly developed BLIs, there are diazabicyclooctane (DBO) molecules, namely zidebactam, durlobactam, and nacubactam, that efficiently inhibit most class A and C (also some class D for zidebactam and durlobactam) β-lactamases, and additionally, they exhibit effective antibacterial activity by targeting PBP-2, in contrast to the presently available DBO avibactam, which have a weaker antibacterial activity. Another class of inhibitors corresponds to the boronic acid derivatives, with taniborbactam inhibiting the hydrolytic activity of class A and C β-lactamases, but also that of metallo-beta-lactamases (MBLs). Taniborbactam inhibits most NDM- and VIM-type β-lactamases (except NDM-9, NDM-30, and VIM-83), but does not show significant inhibitory activity against IMP-type enzymes [15–18]. Altogether, the development of all those new BLIs has promoted the development and the evaluation of novel BL/BLI combinations including aztreonam-avibactam, cefepime-zidebactam (WCK 5107, DBO), cefepime-taniborbactam (VNRX-5133, boronate), meropenem-nacubactam (FPI-1465, DBO), and sulbactam-durlobactam (ETX-2514) which have been tested in several in vitro studies and are now undergoing clinical evaluations, while imipenem-relebactam and meropenem-vaborbactam are already clinically-available [19–27].

The objective of our study was therefore to evaluate the in vitro activity of these new combos against multidrug-resistant Gram-negative isolates showing reduced susceptibility to cefiderocol, and to evaluate the potential benefit of adding a BLI (avibactam, relebactam, nacubactam, zidebactam, or taniborbactam) together with cefiderocol.

Material and methods

Results

Discussion

Our data highlighted that the best in vitro activities against Enterobacterales showing reduced susceptibility or resistance to cefiderocol are cefepime-zidebactam and meropenem-nacubactam, even against E. coli strains exhibiting PBP3 modifications. In addition, MIC values of cefiderocol combined with BLIs, especially zidebactam, nacubactam, and taniborbactam, were significantly lower than those of cefiderocol alone. Those data might be explained by several reasons. First, the reduced susceptibility to cefiderocol in Enterobacterales was mainly associated to the co-production of CMY-like and/or SHV-like β-lactamases in addition to NDM-like enzymes, the former having been already shown to contribute to cefiderocol resistance [8]. Hence, combining cefiderocol with a BLI that can antagonize the activity of those CMY- or SHV-type β-lactamases basically constitute a significant advantage [8, 9]. Second, zidebactam and nacubactam have not only the ability to inhibit class A β-lactamases, but also possess a significant antibacterial activity by targeting the PBP-2 of Enterobacterales; therefore, those BLIs exhibit a so-called enhancer activity, in line with the low MIC values observed in this study and another [30]. Third, our collection included a high proportion of PBP3-modified E. coli strains, that latter feature being known to affect the susceptibility to cefiderocol, but also that of aztreonam-avibactam and cefepime-taniborbactam, considering that cefiderocol, aztreonam, and cefepime mainly act by targeting the PBP-3 [22, 31–33]. Of note, imipenem-relebactam and meropenem-vaborbactam did not show any significant antibacterial activity against the Enterobacterales tested, which can be explained by the majority being MBL producers.

When considering cefiderocol-resistant P. aeruginosa isolates, the most efficient option was imipenem-relebactam, likely due a high proportion of AmpC overproducers and/or co-producers of SHV-like or VEB-like ESBLs. These results are in line with other studies reporting that the imipenem-relebactam combination is one of the best alternatives to treat infections caused by imipenem-non susceptible P. aeruginosa, mainly driven by multiple resistances mechanisms including combinations of OprD inactivation and AmpC or/and efflux overexproduction [34]. Noteworthy, avibactam and vaborbactam have a negligible effect on restoring susceptibility to aztreonam and meropenem, respectively, probably related to the fact that both β-lactams are very good substrates of the MexAB-OprM efflux system, which is commonly overexpressed in a large proportion of clinical isolates and against which the addition of β-lactamase inhibitors is ineffective. Interestingly, the β-lactamase inhibitor relebactam not only showed an excellent inhibitory activity against class A β-lactamases (i.e., SHV-like and VEB-like enzymes), but also against class C β-lactamases such as the intrinsic PDC-like enzymes. Hence, the association of relebactam with imipenem could restore low MIC values against isogenic imipenem non-susceptible P. aeruginosa isolates producing a wide range of non-enzymatic resistance mechanisms identified in that species, eventually contributing to cefiderocol resistance [34, 35]. On the other hand, the cefepime-taniborbactam and cefepime-zidebactam combinations were not as effective against P. aeruginosa isolates as they were against Enterobacterales. This may be explained by the fact that cefepime is particularly affected by the overexproduction of efflux pumps such as MexAB-OprM, or MexXY. In addition, we did not analyze the presence or absence of bla_OXA-like in our P. aeruginosa isolate collection, and it may be that zidebactam and taniborbactam are not efficient β-lactamase inhibitors against OXA-2- or OXA-10-type β-lactamases, which are known to be widely distributed in P. aeruginosa and contributing to the reduced susceptibility to cefiderocol [11, 19]. Finally, zidebactam and nacubactam have a lower intrinsic antibacterial activity against P. aeruginosa in comparison to Enterobacterales, considering that the MIC values are approximately around 4–16 mg/L for zidebactam, and above 32 mg/L for nacubactam for Pseudomonas spp. [36, 37].

When considering cefiderocol-resistant A. baumannii, the main resistance mechanism is driven by the production of PER-like or NDM-like enzymes, as already described [9]. Our data showed that aztreonam-avibactam was not an interesting option, since A. baumannii naturally exhibits high MIC values of aztreonam, and this β-lactam is also hydrolyzed at high level by PER-like enzymes, often produced in that species [9, 17]. Additionally, the class D β-lactamase OXA-23 (which is very commonly produced in carbapenem-resistant isolates) significantly hydrolyzes imipenem and meropenem, but neither nacubactam nor relebactam inhibit OXA-23-like enzymes, eventually leading to resistance to meropenem-nacubactam and imipenem-relebactam. However, durlobactam inhibits class A β-lactamases, such as PER-like enzymes, as well as some class D β-lactamases such as OXA-23, which explains the high susceptibility rate observed for sulbactam-durlobactam in this strain collection. Cefepime being a very good substrate for PER-like enzyme, but also for efflux pumps oftenly overproduced in A. baumannii, this may explain why combinations including taniborbactam and zidebactam were not sufficiently active to restore MIC values in the susceptibility range. Finally, it is worth highlighting that zidebactam and nacubactam do not possess significant direct antibacterial activity in that species [36, 37]. The main feature to be highlighted is that combining cefiderocol with most β-lactamase inhibitors in this study resulted in low MIC values for most isolates. This is most likely due to the ability of all BLI tested to inhibit PER-like enzymes, which were the most relevant resistance factors of cefiderocol detected in this specie. Those results strongly suggest that combination therapies including cefiderocol with a β-lactamase inhibitor, and sulbactam-durlobactam, might be extremely effective in the treatment of infections caused by cefiderocol-resistant A. baumannii isolates.

In conclusion, we showed here that cefepime-zidebactam, meropenem-nacubactam, and aztreonam-avibactam are the best therapeutic alternatives against multidrug-resistant Enterobacterales exhibiting reduced susceptibility or resistance to cefiderocol. By contrast, imipenem-relebactam was the best option against P. aeruginosa isolates. Worryingly, with the exception of sulbactam-durlobactam, none of the novel β-lactam/β-lactamase inhibitor combinations were effective against A. baumannii isolates. Nevertheless, combinations made of cefiderocol and several β-lactamase inhibitors (namely avibactam, taniborbactam, relebectam, zidebactam, and nacubactam) constitute interesting alternative therapeutics against all tested Gram-negative clinical isolates showing reduced susceptibility to cefiderocol.

Code availability

Author contribution

CLT, PN, LP: conceptualization, methodology, and design of the study; CLT, SF: investigation; PN: supervision and funding acquisition; all authors: analysis and interpretation of the results; CLT, PN, LP: writing—original and final draft.

Funding

Open access funding provided by University of Fribourg. This work was financed by the University of Fribourg, Switzerland, the NARA, and by the Swiss National Science Foundation (grant FNS 310030_1888801).

Data availability

All data generated through this study can be available upon request.

Declarations

Footnotes

References