1. Introduction

Multi-drug resistant (MDR) infections pose a significant challenge in healthcare and can spread rapidly in hospital settings.¹² The rise of carbapenem-resistant strains is particularly concerning.³ To address this issue, a variety of strategies are available. Research has shown that targeted combinations of treatments can effectively overcome resistance and improve therapeutic outcomes. In addition, clinical trials are increasingly exploring combinations of antibiotics with other drugs and innovative antibacterial agents in bi-therapy and tri-therapy approaches, with the goal of improving interventions against MDR infections.⁴

    Beta-lactams and beta-lactamase inhibitors have been utilized in combination for many years to treat infections.⁵⁶ Beta-lactams specifically target the final stages of peptidoglycan synthesis, particularly the transpeptidation step. Peptidoglycan is an essential component of the cell wall in most prokaryotes and is not present in eukaryotic cells.Beta-lactam antibiotics are also generally considered safe for use during pregnancy and are not associated with major fetal harm.⁷ Given these factors, beta-lactam antibiotics, when used in conjunction with other compounds, are a reasonable option in treatment regimens for MDR infections.⁸⁹

    Due to the urgent need for effective treatments against MDR isolates, researchers have explored combinations of antibiotics with other drugs or compounds. Numerous studies have demonstrated that certain combinations can reverse specific resistance traits.¹⁰ To develop effective therapeutic strategies for infections caused by MDR isolates, it is essential to implement sophisticated approaches that prevent antibiotic inactivation, including strategies that reduce antibiotic degradation.¹¹

    Beta-lactam/beta-lactamase inhibitor combinations can provide synergistic activity by preventing the degradation of beta-lactam antibiotics by beta-lactamases. However, clinical effectiveness can be compromised by the emergence of inhibitor-resistant beta-lactamases.¹² For example, cilastatin sodium (PubChem CID 23663403), a sodium thioether salt, inhibits renal dehydropeptidase I, the enzyme responsible for imipenem degradation. Imipenem is therefore co-administered with cilastatin to reduce renal metabolism of imipenem, and relebactam can be added as a beta-lactamase inhibitor to improve activity against selected beta-lactamase-producing organisms.¹³¹⁴ Some studies have also explored broader applications of beta-lactamase inhibitors. For instance, clavulanic acid, a beta-lactamase inhibitor used alongside amoxicillin, has been investigated for the treatment of Helicobacter pylori infection.¹⁵

    Beta-lactam antibiotics are effective; however, microorganisms have developed resistance mechanisms against them. Most MDR isolates associated with hospital-acquired infections are resistant to beta-lactam antibiotics, including some beta-lactam/beta-lactamase inhibitor combinations. Despite this, combining these antibiotics with other drugs or compounds can yield synergistic effects.Masoudi et al.¹⁶ reported that certain nanoantibiotics, such as titanium dioxide and zinc oxide nanoparticles, when used in conjunction with amoxicillin-clavulanic acid, can inhibit biofilm formation by MDR Acinetobacter baumannii and Escherichia coli.

    Natural compounds have been used for many years to treat a variety of diseases. Today, numerous natural compounds with antibacterial, antiviral, and antifungal properties have been identified.¹⁰ Certain environmental organisms are significant sources of compounds, particularly secondary metabolites with antibacterial activity.¹⁷ As with other medications, it is important to explore the effects of combining natural extracts with antibiotics.¹⁸-²⁰ These combinations may be synergistic, antagonistic, or indifferent.In this context, we are particularly interested in investigating the synergistic effects of combining natural compounds with beta-lactam antibiotics. Streptomyces species are a well-known source of antibiotics and related compounds, including beta-lactams (e.g., cephalosporins, carbapenems, and monobactams) and beta-lactamase inhibitors such as clavulanic acid and olivanic acid.¹⁷²¹

    Research investigating combinations of antibiotics and natural compounds has reported promising results against bacterial species linked to periodontal disease. An in vitro study by Saquib et al.²² examined the combined effects of three plant extracts and four antibiotics against four bacterial species associated with periodontal disease. The most significant synergistic effect was observed for Punica granatum combined with amoxicillin against Aggregatibacter actinomycetemcomitans, a Gram-negative facultative anaerobe commonly found in the oral microbiota.

    The present review examines crystal structures of beta-lactam/beta-lactamase inhibitor combinations available in the Protein Data Bank (PDB; https://www.rcsb.org). The review is organized into several sections: an overview of beta-lactam/beta-lactamase inhibitor combinations; a summary of European Committee on Antimicrobial Susceptibility Testing (EUCAST; https://www.eucast.org/) and Clinical and Laboratory Standards Institute (CLSI; https://clsi.org/) guidance relevant to laboratory interpretation; and a detailed analysis of available co-crystal structures involving beta-lactams and their binding partners, including approved therapies and newer compounds under investigation.

2.Beta-lactam/beta-lactamase inhibitor combinations

Clinical laboratories depend on standardized guidelines to report resistance phenotypes of bacterial isolates. In this context, we highlight the most recent EUCAST and CLSI recommendations, including expert rules relevant to beta-lactam/beta-lactamase inhibitor combinations.

2.1. EUCAST expert rules

Antibiotics that inhibit bacterial cell wall synthesis, particularly peptidoglycan synthesis, have a high therapeutic index because peptidoglycan is not present in eukaryotic cells.

    Beta-lactam/beta-lactamase inhibitor combinations are included in EUCAST Version 15.0 (2025) (Table 1). Penicillin-beta-lactamase inhibitor combinations are active against many Gram-negative bacteria and some anaerobic Gram-positive species. Cephalosporin-beta-lactamase inhibitor combinations are active against Gram-negative bacilli and Haemophilus influenzae. Monobactam-beta-lactamase inhibitor combinations are used primarily against Enterobacterales. EUCAST Version 15.0 (2025) includes four penicillin-beta-lactamase inhibitor combinations, three cephalosporin-beta-lactamase inhibitor combinations, two carbapenem-beta-lactamase inhibitor combinations, and one monobactam-beta-lactamase inhibitor combination.

2.2. The CLSI expert rules

The CLSI M100 performance standards for antimicrobial susceptibility testing are an important standard used in clinical laboratories. Each year, CLSI provides updated guidance and new additions needed for interpreting antibiogram results obtained by disk diffusion or dilution methods.

    Several beta-lactam/beta-lactamase inhibitor combinations were added relatively recently to CLSI standards (Table 2). In 2024, CLSI included sulbactam-durlobactam. The European Medicines Agency issued a decision (EMEA-002807-PIP01-20-M01; 2022) related to the pediatric investigation plan for durlobactam/sulbactam (https://www.ema.europa.eu/en/medicines/human/paediatric-investigation-plans/emea-002807-pip01-20-m01). Sulbactam-durlobactam is a beta-lactam/beta-lactamase inhibitor combination with a narrow application, targeting the Acinetobacter baumannii-calcoaceticus complex. Its main therapeutic indication is hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia in adults.²³

2.3. Bacterial beta-lactamases

Beta-lactamases (E.C. 3.5.2.6) are enzymes that hydrolyze beta-lactam antibiotics and may show different specificities for penicillins, cephalosporins, and carbapenems. Beta-lactamases are commonly classified by the Ambler molecular classification (classes A-D); in addition, standardized residue-numbering schemes (e.g., Ambler/ABL numbering) are used to compare homologous enzymes.²⁴-²⁶ The Bush-Jacoby-Medeiros functional classification offers an additional approach based on substrate and inhibitor profiles (Table 3).²⁷

    One notable group is class B beta-lactamases, also known as metallo-beta-lactamases (MBLs), which require zinc for catalytic activity. Many beta-lactamases are produced by human pathogens, and these enzymes continue to evolve, making antibiotic-resistant infections increasingly challenging to treat. As a result, functional groupings are updated as new enzymes emerge and as additional information becomes available on hydrolysis spectra and inhibitor susceptibility.²⁸

2.4. Beta-lactamase inhibitors

Beta-lactamase inhibitors, when used in combination with beta-lactam antibiotics, can improve activity against beta-lactamase-producing bacteria. Table 4 summarizes key characteristics of beta-lactamase inhibitors approved for clinical use. Notably, six of the seven approved beta-lactamase inhibitors contain sulfur in their chemical structure.

    Clavulanic acid is a conjugate acid of clavulanate produced by fermentation of Streptomyces clavuligerus.²⁹ It has weak antibacterial activity alone and is used in combination with penicillins such as amoxicillin and ticarcillin. Sulbactam, tazobactam, and enmetazobactam are penicillanic acid sulfones.

    Sulbactam is a beta-lactamase inhibitor and can also bind penicillin-binding proteins (PBPs) in Gram-positive and Gram-negative species.³⁰ Durlobactam is a diazabicyclooctane beta-lactamase inhibitor (PubChem CID 89851852) administered in combination with sulbactam (Food and Drug Administration [FDA] approval in 2023). This inhibitor targets serine beta-lactamases (SBLs; class A, C, and D) and is indicated for hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia caused by susceptible isolates of the A. baumannii-calcoaceticus complex.³¹

    Tazobactam is a beta-lactamase inhibitor with activity against the OHIO-1, SHV-1, and TEM groups of beta-lactamases.³² It was approved by the FDA in 1994 in combination with piperacillin for infections caused by Gram-positive and Gram-negative species, including anaerobes. Ceftolozane-tazobactam was initially approved in 2014, and its indication was expanded in 2019 to include hospital-acquired bacterial pneumonia and ventilator-associated pneumonia with Gram-negative susceptible isolates. In combination with metronidazole, it can be used for complicated intra-abdominal infections.³³

    Avibactam is a member of the class of azabicycloalkanes approved by the FDA in 2015 in combination with ceftazidime for the treatment of complicated urinary infections (including pyelonephritis) caused by susceptible microorganisms, including E. coli, Klebsiella pneumoniae, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Proteus spp., and Pseudomonas aeruginosa. It can also be administered with metronidazole for the treatment of complicated intra-abdominal infections caused by susceptible microorganisms, including E. coli, K. pneumoniae, Proteus mirabilis, Providencia stuartii, E. cloacae, Klebsiella oxytoca, and P. aeruginosa.³⁴,³⁵

    Relebactam is a derivative of diazabicyclooctane, approved by the FDA in 2019 in combination with imipenem and cilastatin. It inhibits Class A and Class C serine beta-lactamases (SBLs) by forming a covalent acyl-enzyme intermediate with the active-site serine residue.³⁶

    Vaborbactam is a cyclic boronic acid pharmacophore derivative approved by the FDA in 2017 in combination with meropenem for the treatment of complicated urinary tract infections (including pyelonephritis) caused by susceptible microorganisms, including E. coli, K. pneumoniae, and E. cloacae species complex.³⁷ A triple-combination strategy (e.g., meropenem-vaborbactam-aztreonam) has been proposed for selected resistant phenotypes, including K. pneumoniae isolates resistant to ceftazidime-avibactam.³⁸

    Enmetazobactam is a penicillanic acid sulfone derivative that was approved by the FDA on February 23, 2024, in combination with cefepime for the treatment of complicated urinary tract infections.³⁹

    Efforts to address inhibitor-resistant infections have prompted extensive research focused on developing new beta-lactamase inhibitors. Table 5 summarizes several investigational compounds with activity against serine beta-lactamases (SBLs) and/or metallo-beta-lactamases (MBLs). X-ray structures of these inhibitors co-crystallized with beta-lactamases are discussed in the following section.

    Taniborbactam is a small molecule and a cyclic boronate derivative currently under investigation in Phase III clinical trials. When combined with cefepime, it exhibits activity comparable to aztreonam-avibactam against serine beta-lactamases (SBLs) and metallo-beta-lactamases (MBLs).⁵⁶

    Xeruborbactam is also a small molecule and a cyclic boronate derivative currently under investigation. It is active against selected SBLs and MBLs.⁴² When combined with meropenem, xeruborbactam shows activity against some imipenemase-producing Enterobacterales; however, meropenem alone is ineffective against imipenemase-producing Pseudomonas aeruginosa.⁴¹

    Nacubactam is a derivative of diazabicyclooctane and serves as an inhibitor of SBLs. It is being investigated for complicated urinary tract infections (ClinicalTrials.gov identifier: NCT03174795; EudraCT: 2021-001396-16). Misawa et al.⁴⁵ reported that nacubactam combined with imipenem and certain beta-lactams (e.g., cefazolin, cefotiam, cefoxitin, or cefuroxime) was effective against Mycobacterium abscessus.

    Zidebactam is another derivative of diazabicyclooctane that has a dual mechanism: It inhibits SBLs and binds to penicillin-binding protein 2 (PBP2).⁴⁶,⁴⁸ It is being investigated in combination with cefepime for infections caused by Gram-negative beta-lactamase-producing organisms.⁴⁹,⁵⁰

3.Beta-lactamases Catalytic Mechanisms

Crystallographic studies can elucidate molecular mechanisms underlying catalysis.

    A complex structure of AmpC beta-lactamase with a series of acylglycine boronic acids provides insights into inhibitor affinity for serine beta-lactamases (SBLs). Caselli et al.⁵⁷ demonstrate that the R1 side chains of beta-lactams can contribute to affinity for beta-lactamases (PDB ID: 1FSY).

    Recent studies highlight the pivotal role of Ser130 in the active site of class A SBLs. X-ray crystal structures of SHV-like beta-lactamases (PDB IDs: 1TDL, 1TDG) indicate that Ser130 is crucial for interactions with beta-lactamase inhibitors such as clavulanic acid and tazobactam.⁵⁸ Site-directed mutagenesis has provided valuable insights into inhibitor-resistant beta-lactamases, revealing that certain residues affect acylation rates for specific substrates. For instance, a CTX-M-14 variant (K234R) from E. coli showed that substitution of Lys234 with arginine decreases the acylation rate of cefotaxime. Furthermore, the conformational change associated with residue 234 reduces the acylation of clavulanic acid (PDB ID: 7K2X).⁵⁹ The K234R substitution in SHV-1 beta-lactamase can be addressed by derivatives such as SA2-13 ((3R)-4-[(4-carboxybutanoyl)oxy]-N-(1E)-3-sulfino-D-valine) (PDB ID: 4MBK).⁶⁰

    Mutations in the active site of beta-lactamases can affect enzyme stability. However, mutations located outside the active site can also stabilize mutated enzymes. For instance, the M182T mutation in TEM-64 beta-lactamase (PDB ID: 1JWZ) illustrates this effect.⁶¹

4.Crystal Structures of Beta-lactams and Inhibitor-resistant Beta-lactamases

A search of the Protein Data Bank (PDB) identified numerous beta-lactamase structures, including complexes co-crystallized with inhibitors, as well as enzymes involved in beta-lactam antibiotic biosynthesis. Many multidrug-resistant (MDR) isolates carry inhibitor-resistant beta-lactamases, which can reduce the effectiveness of beta-lactam/beta-lactamase inhibitor combinations. Structural analysis of these inhibitor-resistant beta-lactamases can clarify the molecular basis of resistance.

    Using the advanced search query “inhibitor-resistant” AND “beta-lactamase,” 124 structures were retrieved from the PDB database on August 11, 2025. After manual curation, 35 entries corresponded to beta-lactamase structures co-crystallized with inhibitors. Additionally, five entries corresponded to penicillin-binding proteins (PBPs) co-crystallized with inhibitors. Therefore, we included 35 beta-lactamase structures and five PBP structures in the present analysis.Sequence analysis and multiple alignment can provide additional insights relevant to inhibitor-resistant beta-lactamases. The effectiveness of beta-lactamase inhibitors extends to both Gram-negative and Gram-positive bacterial species.

4.1. Beta-lactamase Inhibitors Effective Against Beta-lactamases from Gram-negative Bacteria

Beta-lactamases can be broadly grouped by active-site chemistry into serine beta-lactamases (SBLs) and metallo-beta-lactamases (MBLs).

4.1.1. SBLs with Beta-lactamase Inhibitor Crystal Structures

Many inhibitor-resistant beta-lactamases are variants of broad-spectrum enzymes.⁶² One of the most studied SBLs is the TEM-1 plasmid-encoded class A beta-lactamase, which belongs to the transpeptidase superfamily (UniProt/SwissProt: P14677). The name “TEM” originates from the patient (Temoniera) from whom it was first isolated.⁶³

    The crystal structure of TEM-76 beta-lactamase (PDB ID: 1YT4) revealed a water molecule (Wat1023) in the active site that replaces the side-chain hydroxyl group of Ser130. A similar water molecule that facilitates clavulanate crosslinking has also been observed in other TEM beta-lactamases, including TEM-32 and TEM-84.⁶⁴

    Wang et al.⁶¹ proposed that inhibitors structurally similar to substrates are less likely to select for resistance mutations. To test this hypothesis, they examined a series of analogs against the TEM-1 class A beta-lactamase. X-ray crystallography and kinetic experiments showed that some boronic analogs mimic the transition states of acylation and deacylation (PDB IDs: 1NYY, 1NXY, and 1JWZ) (see Figure 1).⁶⁵ The 3D structure of TEM-64 (PDB ID: 1JWZ) was downloaded from the PDB (https://www.rcsb.org/structure). Interactions were visualized using BIOVIA Discovery Studio Modeling Environment (Release 2017; Dassault Systèmes, USA; http://accelrys.com).

    CTX-M is a class A beta-lactamase that was first identified in a cefotaxime-resistant E. coli isolated from an ear sample of a newborn.⁶⁶ Unlike the plasmidic CTX-M, the SHV-like beta-lactamase (sulphydryl variable) likely originated from a chromosomal penicillinase found in Klebsiella pneumoniae.⁶⁷Studies by Winkler et al.⁶⁸ and Soeung et al.⁵⁹ showed that an arginine residue interacts with clavulanic acid in the class A beta-lactamases SHV from K. pneumoniae and CTX-M from E. coli. The structure of the mutated SHV-1 K234R (PDB IDs: 4FCF and 7K2W) indicates that two specific residues, Arg234 and the movement of Ser130, are responsible for resistance to clavulanate. Furthermore, Soeung et al.⁵⁹ suggest a distinction between penicillins and cephalosporins, recommending that coupling clavulanic acid with cephalosporins should be prioritized to reduce the development of inhibitor resistance (PDB IDs: 7K2Y and 7K2W).The importance of Ser130 has led to the development of new inhibitors such as SA-13, which are effective against SHV mutants from K. pneumoniae (PDB IDs: 3V50, 3V5M) (see Figure 2).⁶⁹

    Combination (“tri-therapy”) strategies are used clinically, and structural studies can help clarify effective combinations. The crystal structure of the serine-class A beta-lactamase from Stenotrophomonas maltophilia (PDB IDs: 5NE1 and 5NE3) reveals that ceftazidime, avibactam, and aztreonam work synergistically. This synergy arises because SBLs do not interact with aztreonam as substrates, in contrast to MBLs.⁷⁰

4.1.2. MBLs with Beta-lactamase Inhibitor Crystal Structures

Liu et al.⁴⁰ demonstrated that taniborbactam (VNRX-5133), a boronic-acid-containing broad-spectrum beta-lactamase inhibitor, can restore the efficacy of beta-lactams against carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacterales (PDB IDs: 6SP6 and 6SP7).Additionally, several triazole-based compounds have shown promise for inhibiting broad-spectrum beta-lactamases based on molecular modeling and in vitro enzymatic studies. Bersani et al.⁴³ identified three 1,2,4-triazole-3-thione derivatives that are effective against NDM-1 (New Delhi MBL), VIM-2 (Verona integron-encoded MBL), and VIM-4 MBLs (PDB IDs: 8B1W, 8B1Z, and 8B20).

    Furthermore, Davies et al.⁵² identified new diazabicyclooctanes, including ANT3310 and ANT90, that are effective against serine beta-lactamases (SBLs). Notably, ANT3310 restores carbapenem activity not only against carbapenem-resistant Enterobacterales but also against OXA-type beta-lactamase (OXA)–carrying Acinetobacter baumannii isolates (PDB ID: 6ZXI).Compared to relebactam and avibactam, derivatives of ANT3310 contain a fluorine substituent, which may contribute to enhanced efficacy (see Figure 3). OXA-type beta-lactamases are plasmid-mediated enzymes particularly resistant to oxacillin.

    Crystal structures help clarify the interactions of newer beta-lactamase inhibitors compared with older agents. Figure 4 shows interacting residues of OXA-48 from Klebsiella pneumoniae (PDB ID: 6ZXI) with the new diazabicyclooctane ANT3310.

    Leiris et al.⁵³ designed and optimized a series of pyridine-2-carboxylic acid derivatives suitable for inhibition of MBL-producing isolates (NDM, VIM, and IMP). X-ray structures of VIM-2 carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa with these new inhibitors (PDB IDs: 5MXQ, 5MXR, 6HF5) show inhibitor interactions with residues from the so-called “Zn²⁺” site (Asp, Cys, His).

    The compound ETX2514 (PubChem CID: 137349620), in combination with sulbactam, is a potent inhibitor targeting the class D OXA-24 from Acinetobacter baumannii (PDB ID: 5VFD).⁵⁵

    Crystallographic studies, along with thermodynamic experiments, identified a 2-triazolylthioacetamide derivative that interacts with key residues in the active site of VIM-2 MBL from P. aeruginosa (PDB ID: 6KW1). The inhibitor binds to Zn²⁺ ions and interacts with Asn233, His263, and a water molecule (see Figure 5).⁵⁴

4.2. PBPs with Inhibitor Co-crystal Structures

To combat antibiotic resistance in bacterial isolates producing beta-lactamases, one strategy is to target additional components of cell-wall synthesis. Penicillin-binding proteins (PBPs), which are found in both Gram-negative and Gram-positive bacteria, play a crucial role in peptidoglycan synthesis.Goldberg et al.⁷¹ described a novel γ-lactam siderophore (YU253911 and YU253434) that binds to Pseudomonas aeruginosa PBP3 through multiple hydrogen bonds at the active site (PDB IDs: 7LC4 and 6VOT). Additionally, the γ-lactam siderophore showed synergy with sulbactam against multidrug-resistant Acinetobacter spp. in an animal model.⁷²

    Meticillin-resistant Staphylococcus aureus (MRSA) expresses a PBP, PBP2a, which supports cell-wall synthesis in the presence of beta-lactam antibiotics. Quinazolinone binds to an allosteric site and can facilitate beta-lactam binding. When combined with tazobactam, this interaction demonstrated synergy in an animal model (PDB IDs: 6Q9N and 6H5O).⁷³

    A recent study reported that specific boronate inhibitors are effective against multidrug-resistant Neisseria gonorrhoeae. X-ray crystal structure analysis indicates that the VNRX-14079 boronate compound binds to the transpeptidase domain of PBP2 from N. gonorrhoeae (PDB IDs: 9MCZ and 9MD0).⁷⁴ This interaction involves a covalent bond between the boron atom and the active-site serine residue at position 310, as well as interactions involving the β3–β4 loop. These findings highlight the potential of these inhibitors in addressing this critical public health challenge.

4.3. Beta-lactamase inhibitors effective against antibiotic-resistant Gram-positive species

Recent in silico studies have screened and identified benzimidazole-based compounds with the potential to restore beta-lactam susceptibility in MRSA. Nguyen et al.¹⁵ determined the X-ray structure of the S. aureus BlaR1 regulatory/sensor protein (UniProt P18357) crystallized with two benzimidazole-based compounds. The first inhibitor, a boronate compound (boronate 4), is [1-[[2,4-bis(trifluoromethyl)phenyl]methyl]benzimidazol-2-yl]sulfanylmethyl-{3}-oxidanylbis(oxidanyl)boron] (PDB ID: 8C0P). The second inhibitor is an imidazole compound, 3-[[2,4-bis(trifluoromethyl)phenyl]methyl]-5-(hydroxymethyl)-1H-imidazole-2-thione (PDB ID: 8C0S). Boronate 4 inhibitor interacts with the active-site serine residue and shows efficacy in animal experiments when combined with oxacillin or meropenem against MRSA (Figure 6).

    Caselli et al.¹⁷ reported that two acylglycine boronic acids showed synergy with amoxicillin against S. aureus expressing a Group 2 beta-lactamase.

    Mycobacterium tuberculosis is commonly classified as a high-G+C Gram-positive bacterium; however, it stains poorly with the Gram stain because of its lipid-rich cell envelope (notably mycolic acids), which underlies its acid-fast property. Some genome-based analyses have suggested that M. tuberculosis shares more features with Gram-negative bacteria than with typical Gram-positive bacteria.¹⁶ BlaC is the principal beta-lactamase of M. tuberculosis. Resistance to beta-lactam/beta-lactamase inhibitor combinations is considered unlikely to arise primarily through structural alterations of BlaC. Kurz et al.¹⁷ demonstrated that an inhibitor-resistant BlaC variant retained susceptibility to meropenem-clavulanic acid combination (PDB ID: 4JLF).

5.Discussion

Multidrug-resistant organisms are a major global concern. There is an urgent need for alternatives to existing treatments for these MDR strains, including new drug combinations. However, developing new compounds is a lengthy process. To address the critical need for effective treatments against MDR clinical isolates, researchers are actively exploring innovative solutions that combine antibiotics with other drugs or compounds. Advances in in silico studies have significantly improved the selection and optimization of compounds that may help reverse certain antibiotic-resistant traits.¹⁵ Artificial intelligence (AI) tools also show promise for improving the design of new inhibitors.

    Infections caused by MDR organisms and the continued evolution of beta-lactamases challenge healthcare professionals to integrate clinical needs with basic science. Clinical laboratories must keep pace with newly approved synergistic combination treatments for infections caused by beta-lactamase-producing isolates. Accordingly, expert guidelines from EUCAST and CLSI support the interpretation of antibiogram results and help ensure alignment with current standards. However, both established therapies and new drugs must be continuously reviewed in light of beta-lactamase evolution and the ongoing emergence of new mutations.

    The prevalence of beta-lactamases in Gram-negative bacilli, particularly within the order Enterobacterales, is a major concern. The TEM family of beta-lactamases has been studied extensively. TEM-1-type beta-lactamases are encoded by a gene located within the transposon Tn3. The association of this gene with plasmids is crucial because it facilitates rapid and widespread transmission of antibiotic resistance. Furthermore, mutations within the active-site cavity may be accompanied by secondary mutations at positions distant from the active site, which can help restore structural stability of the enzyme.

    Understanding the molecular factors driving the evolution of inhibitor-resistant beta-lactamases is essential for designing new antibacterial compounds and for re-evaluating synergistic combinations. The significance of Ser130 in the active site of SBLs has supported the development of new inhibitors, such as SA-13, which retain activity against selected SHV mutants. These inhibitors may help address challenges posed by SHV mutations and improve treatment efficacy. Key structural information is available in PDB IDs 3V50 and 3V5M, which describe interactions relevant to inhibitor activity against resistant variants.⁶⁹

    X-ray structures of intermediate forms of SHV-1 mutants in complex with inhibitors provide insight into conformational changes in the inhibitor-enzyme complex (PDB IDs: 2H10, 2H0Y, and 2H0T).¹⁸ In addition, boronic acid inhibitors have been evaluated as a strategy to address inhibitor-resistant beta-lactamases.¹⁶

    The emergence of inhibitor-resistant beta-lactamases is reducing treatment options and increasing the need for new synergistic antibiotic combinations. Bi-therapy and tri-therapy strategies offer potential approaches for resistant infections. Although beta-lactam/beta-lactamase inhibitor combinations have been used for many years, the rapid emergence of beta-lactamases resistant to currently available inhibitors continues to complicate the treatment of infections caused by MDR isolates. As a result, tri-therapy is increasingly considered, especially for nosocomial infections. A combination of imipenem, cilastatin, and relebactam has shown activity against imipenem-non-susceptible Gram-negative pathogens.¹³¹⁴ In addition, quinazolinone combined with piperacillin–tazobactam has demonstrated efficacy against MRSA.⁷³

    X-ray structures of beta-lactamases in complex with inhibitors provide valuable insights relevant to inhibitor design and resistance mitigation. However, structural findings should be complemented by kinetic studies assessing acylation and deacylation rates. Promising directions include rational drug design targeting broad-spectrum MBLs and SBLs. In this context, AI may accelerate structure-based inhibitor design.

6.Conclusion

The treatment of MDR infections often presents significant challenges because of limited therapeutic options, which can necessitate bi-therapy or tri-therapy. Investigation of synergistic combinations of beta-lactams and beta-lactamase inhibitors is particularly promising because these therapies target peptidoglycan, which is exclusively found in prokaryotes. However, the emergence of beta-lactamases resistant to inhibitors highlights the need to optimize both existing and new inhibitors. X-ray crystal structures available in the PDB are valuable for understanding drug interactions and for comparing new compounds with approved inhibitors. Focusing research on specific drug combinations can support progress through coordinated efforts in both research and clinical settings.

Mihaela Ileana Ionescu is a Guest Editor of this special issue. The author declared that she has no known competing financial interests or personal relationships that could have influenced the work reported in this paper.