Advertisement View all journals Search Login Explore content About the journal Publish with us Sign up for alerts RSS feed nature nature reviews microbiology review articles article Review Article Published: 05 March 2019 Interplay between β-lactamases and new β-lactamase inhibitors Karen Bush & Patricia A. Bradford Nature Reviews Microbiology 17, 295–306 (2019)Cite this article 9569 Accesses 158 Citations 64 Altmetric Metrics An Author Correction to this article was published on 29 May 2019 A Publisher Correction to this article was published on 30 April 2019 This article has been updated Abstract Resistance to β-lactam antibiotics in Gram-negative bacteria is commonly associated with production of β-lactamases, including extended-spectrum β-lactamases (ESBLs) and carbapenemases belonging to different molecular classes: those with a catalytically active serine and those with at least one active-site Zn2+ to facilitate hydrolysis. To counteract the hydrolytic activity of these enzymes, combinations of a β-lactam with a β-lactamase inhibitor (BLI) have been clinically successful. However, some β-lactam–BLI combinations have lost their effectiveness against prevalent Gram-negative pathogens that produce ESBLs, carbapenemases or multiple β-lactamases in the same organism. In this Review, descriptions are provided for medically relevant β-lactamase families and various BLI combinations that have been developed or are under development. Recently approved inhibitor combinations include the inhibitors avibactam and vaborbactam of the diazabicyclooctanone and boronic acid inhibitor classes, respectively, as new scaffolds for future inhibitor design. Introduction β-Lactam antibiotics are the most frequently prescribed antibacterial agents in the hospital setting due to their safety, efficacy and breadth of spectrum of activity1. The four main classes of β-lactams include penicillins, cephalosporins, carbapenems and monobactams, all of which include in their structure a four-membered azetidinone ring (Fig. 1). Although multiple iterations of the first three have been introduced into clinical practice, only a single monobactam, aztreonam, has been approved by the FDA1. All these agents have a common bacterial killing mechanism that involves the inactivation of transpeptidases that are required for the terminal step of bacterial cell wall synthesis2. The penicillin-binding proteins (PBPs), which include these transpeptidases, all have a serine at the enzymatic active site that is acylated by the β-lactams in a reaction that is essentially irreversible within the lifetime of a bacterial cell3. This reaction is illustrated in Fig. 2, whereby PBPs undergo rapid acylation (E–BL) followed by slow deacylation to regenerate active enzyme (E) and a hydrolysed, ineffective ring-opened β-lactam (BL*). Fig. 1: Structures of β-lactams and β-lactamase inhibitors. a | Generic structures of the β-lactams penicillin, cephalosporin, carbapenem and monobactam are shown. b | Depicted are the structures of clavam and the sulfone β-lactamase inhibitors clavulanic acid, sulbactam, tazobactam and enmetazobactam (formerly AAI101). c | Structures of the diazabicyclo[3.2.1]octanone (DBO) β-lactamase inhibitors avibactam, relebactam, nacubactam, zidebactam and ETX2514 are shown. d | Shown are the structures of serine β-lactamase inhibitors vaborbactam and VNRX-5133, which are derived from boronic acid. e | The structure of the metallo-β-lactamase inhibitor ANT431 is shown. Full size image Fig. 2: General reaction pathway for the interaction of a β-lactam with penicillin-interactive enzymes. Penicillin-interactive enzymes such as a penicillin-binding protein (PBP; designated as E) or a β-lactamase having a serine residue at the active site (E) bind a β-lactam (BL) to form a reversible complex (E ∙ BL). This complex may proceed to a stable acyl enzyme (E–BL) caused by the reaction of the β-lactam with the active-site serine of the enzyme. Addition of water to the acyl enzyme results in breaking of the β-lactam bond, resulting in a microbiologically inactive antibiotic (BL*). Hydrolysis of the acyl enzyme occurs rapidly for a β-lactamase and slowly for a PBP. Full size image Arguably, the biggest challenge to the use of the β-lactams is the presence of β-lactamases1, which are hydrolysing bacterial enzymes that facilitate the rapid addition of a molecule of water across the common β-lactam bond, rendering the antibiotic ineffective and incapable of inhibiting the PBPs (Fig. 2). β-Lactamases contain either a reactive serine residue in their active site (serine β-lactamases) or at least one catalytically functional divalent zinc atom (metallo-β-lactamases (MBLs)) (Fig. 3). Serine β-lactamases are diverse in structure and hydrolytic profile; however, all these enzymes hydrolyse β-lactams in a multistep reaction via a serine residue at their catalytic site. Initially, a negatively charged carboxylate, or similarly charged group, on the side chain of the azetidinone ring of the β-lactam antibiotic is attracted to the active site of the β-lactamase by positively charged residues. Once the β-lactam is properly positioned, it forms key hydrogen bonds with the β-lactamase4. Next, the β-lactamase is acylated via a conserved serine residue (Ser70 in class A enzymes and Ser64 in class C enzymes; see below) functioning as the reactive nucleophile in this acylation reaction. Subsequently, a water molecule is activated to deacylate the β-lactam–β-lactamase complex, resulting in release of the ring-opened and ineffective antibiotic and regeneration of active β-lactamase. MBLs require at least one and usually two Zn2+ ions, bridged by a hydroxide ion that facilitates nucleophile attack on the carbonyl oxygen atom of the β-lactam ring, leading to hydrolysis and inactivation of the antibiotic (Fig. 3). One Zn2+ is held in place by three histidine residues. Many MBLs contain a second zinc-binding site, with zinc ligands of Asp, Cys and/or His, and His also functioning in the hydrolysis reaction5. Fig. 3: General mechanism of hydrolysis of β-lactams for serine and metallo-β-lactamases. For serine β-lactamases (indicated as E-Ser–OH), a negatively charged carboxylate, or similarly charged group, on the side chain of the azetidinone ring of the β-lactam is attracted to the active site of the β-lactamase by positively charged residues. The β-lactam forms key hydrogen bonds with the β-lactamase, which is then acylated. Subsequently, a water molecule is activated to deacylate the β-lactam–β-lactamase complex, leading to the generation of an active β-lactamase and a ring-opened, microbiologically inactive molecule. Metallo-β-lactamases (MBLs) require at least one and usually two Zn2+ ions, bridged by a hydroxide ion that facilitates nucleophile attack on the carbonyl oxygen atom of the β-lactam ring, leading to hydrolysis and inactivation of the antibiotic. One Zn2+ is held in place by three histidine residues. Many MBLs contain a second zinc-binding site, with zinc ligands of Asp, Cys and/or His, and His also functioning in the hydrolysis reaction. Full size image In the early to mid-1940s, the first clinically relevant reports of resistant bacteria due to production of β-lactamase described staphylococcal penicillinases that hydrolysed penicillin G6,7. After the introduction of ‘penicillinase-stable’ penicillins, the emphasis shifted to β-lactamases in Gram-negative bacteria, including enzymes that are both chromosomally produced, and more recently, plasmid encoded. At this time, more than 2,770 unique, naturally occurring β-lactamases have been documented8. As these enzymes posed a major threat to the therapeutic use of β-lactams for decades, the concept emerged of developing combinations of a β-lactamase inhibitor (BLI) with a microbiologically active penicillin or cephalosporin formulated as a single product9. In the 1980s, the first anti-infective drug combination that did not contain two microbiologically active components was approved by a major regulatory agency; two of the early BLI combinations (amoxicillin–clavulanic acid and piperacillin–tazobactam; Fig. 1b) quickly became some of the most widely prescribed antibacterial agents in recent time. In this Review, the families of β-lactamases that are most important medically are described; various BLI combinations that have been developed to counteract these enzymes, as well as novel investigational agents currently under development, are included. As with all new antimicrobial agents, resistance to all of the approved BLI combinations has been reported, either in vitro or in clinical practice. These mechanisms will be discussed, emphasizing resistance mechanisms selected specifically by the BLI combinations, followed by a short commentary concerning the future of the currently crowded field of BLI combinations. β-Lactamase nomenclature Two major classification schemes are currently used for β-lactamases: the Ambler molecular classification, which separates β-lactamases into distinct molecular classes (A, B, C and D) on the basis of their amino acid sequence, and the Bush–Jacoby–Medeiros functional classification scheme, which categorizes β-lactamases according to similarities of substrate hydrolysis profiles and responses to various inhibitors10,11,12 (Table 1). β-Lactamases of Ambler classes A, C and D are all serine β-lactamases, whereas class B comprises MBLs10. The functional classification scheme categorizes β-lactamases into four groups and multiple major subgroups (groups 1, 2 (with the major subgroups 2a, 2b, 2br, 2d, 2be, 2c and 2f) and 3)11,12. A comparison of the two classification systems is summarized in Table 1. Note that the nomenclature of individual β-lactamases has not been standardized as the names have been designated by the authors who first described them. Novel enzymes were initially named on the basis of random, and sometimes creative, characteristics, ranging from place names to functional descriptions. After DNA sequencing became routine in the 1990s, a new enzyme with high sequence homology to an index enzyme was numbered consecutively as a member of that family. In 2010, Jacoby provided a list of the most common β-lactamase names and their sources13. Table 1 Comparison of two β-lactamase classification schemes Full size table Class A: broad-spectrum serine β-lactamases Ambler class A β-lactamases comprise the largest number of enzymes with a very wide spectrum of activity. Penicillinases that belong to functional group 2b are broad-spectrum enzymes as they hydrolyse the first penicillins and cephalosporins approved for clinical use. Two commonly encountered β-lactamases found in Enterobacteriaceae (designated TEM-1 and SHV-1) belong to this group. Before the emergence of extended-spectrum β-lactamases (ESBLs; see below), TEM-1 was the primary cause of ampicillin resistance in Escherichia coli and remains the highest contributor to penicillin resistance in Neisseria gonorrhoeae, whereas ampicillin resistance in Klebsiella pneumoniae was most often attributed to SHV-1. High expression levels of TEM-1 have been associated with resistance to cephalosporins and BLI combinations14. Extended-spectrum β-lactamases ESBLs belonging to Ambler class A and functional group 2be are serine β-lactamases. They hydrolyse penicillins and cephalosporins that are both narrow and expanded in their microbiological spectrum. Most ESBLs are susceptible to inhibitors that have been approved by the FDA. The first ESBLs that emerged were derivatives of TEM and SHV that had amino acid substitutions resulting in an ’expanded’ substrate profile. The replacement of Gly238 with Ser, Ala or Asp is a frequent mutation in both TEM-type and SHV-type ESBLs4,15. TEM-type and SHV-type ESBLs were commonplace for the first decade of ESBL detection, but in recent years, their importance has diminished due to the worldwide dissemination of CTX-M-producing isolates. First reported in the 1980s, CTX-M-type β-lactamases have spread worldwide and now are the most common ESBLs16. These enzymes can be divided into five distinct major clusters that are phylogenetically related17. In general, CTX-M β-lactamases hydrolyse cefotaxime and ceftriaxone better than ceftazidime18. CTX-M-type enzymes are readily inhibited by tazobactam and by newer BLIs, such as avibactam, relebactam and vaborbactam19,20,21. CTX-M β-lactamases are commonly found in E. coli and K. pneumoniae but have also been found in other species of Enterobacteriaceae, including Salmonella spp., Shigella spp., Citrobacter freundii, Enterobacter spp. and Serratia marcescens as well as some non-fermentative bacteria22,23. Other clinically important ESBL families include GES, PER, VEB, BES, BEL, SFO and TLA, but they are found with much lower prevalence than CTX-M types24,25. Inhibitor-resistant class A β-lactamases Inhibitor-resistant β-lactamases (functional group 2br) are TEM and SHV enzymes that have amino acid substitutions that render the enzyme refractory to inhibition by the BLIs clavulanic acid and sulbactam; however, most remain susceptible to inhibition by tazobactam and avibactam11,12,26,27. Most inhibitor-resistant β-lactamases are variants of the TEM-1 enzyme (also known as inhibitor-resistant TEM) and contain amino acid substitutions at Met69, Ser130, Arg244, Arg275 or Asn276 (ref.28). Interestingly, the overall effect of these mutations caused the enzymes to become less efficient in hydrolysing some penicillins and cephalosporins such as cephalothin29, thereby retaining susceptibility to these agents in the producing organisms30. A few mutants of TEM β-lactamases have been described that maintain the ESBL phenotype but also demonstrate resistance to inhibitors and have been referred to as complex mutant TEM β-lactamases28. Serine carbapenemases Ambler class A enzymes that can hydrolyse carbapenems (serine carbapenemases) are clustered in Bush–Jacoby–Medeiros functional group 2f11. β-Lactamases included in this group are KPC, SME, NMC-A, IMI and certain variants of GES31,32. KPC-2 and KPC-3 variants have the largest distribution of all of the serine carbapenemases worldwide. KPC has been detected mainly in K. pneumoniae but also in many species of Enterobacteriaceae, Acinetobacter spp. and Pseudomonas aeruginosa33. KPC-type enzymes are not inhibited by clavulanate or tazobactam; however, they are potently inhibited by avibactam and vaborbactam21,33. GES enzymes were initially reported as ESBLs, but a single point mutation expanded the spectrum of these enzymes, adding carbapenem-hydrolysing activity to GES-2, GES-4, GES-11 and GES-14 (refs32,34). NMC-A and IMI enzymes are chromosomally encoded carbapenem-hydrolysing β-lactamases found in Enterobacter cloacae35. Similarly, SME is a chromosomal enzyme exclusively from S. marcescens showing greatest amino acid sequence similarity (70%) with NMC-A36. SME enzymes confer resistance to carbapenems, but they have limited hydrolytic activity against extended-spectrum cephalosporins37. Metallo-β-lactamases MBLs, members of Ambler class B and Bush–Jacoby–Medeiros group 3, are distinguished by the presence of Zn2+ at the active site38. MBLs hydrolyse most β-lactams, including carbapenems, but they do not hydrolyse monobactams. They can be inhibited by chelating agents such as EDTA; however, they are not inhibited by inhibitors in the current FDA-approved BLI combinations. Plasmid-encoded MBLs pose an important clinical threat because they are located in mobile genetic structures that can disseminate and harbour resistance genes to other antimicrobial classes31. Among acquired MBLs, IMP, VIM and NDM variants are the most widespread and have now been found worldwide39. They have been identified in various Gram-negative clinical isolates, including several species of Enterobacteriaceae, P. aeruginosa and Acinetobacter spp. IMP enzymes were the first acquired MBLs to be described. IMP-1 was detected in an isolate of P. aeruginosa from Japan in 1988 (ref.40) but is now present in other Gram-negative species39. VIM-1 was described 1999 in a multidrug-resistant P. aeruginosa isolate from Verona, Italy41. VIM-type enzymes have become widespread and are especially prevalent in the Mediterranean region34. VIM variants are often embedded in class 1 integrons. NDM-1 was first described in an isolate of K. pneumoniae from a patient who had previously been admitted to two different hospitals in India during 2007 and then transferred to Sweden, where the organism was identified and characterized42. However, since that time, NDM-type enzymes have spread worldwide due to their inclusion in a complex mobile element. Other MBLs that are less commonly acquired tend to be restricted to certain geographic areas or have been described only in a few instances, including SPM and GIM, which were found in P. aeruginosa isolates from Brazil (Sao Paulo) and Germany, respectively43,44,45,46. AmpC-type β-lactamases AmpC-type β-lactamases belong to Ambler class C (Bush–Jacoby–Medeiros functional group 1)11. They confer resistance to most cephalosporins, including expanded-spectrum cephalosporins such as ceftazidime, cefotaxime and ceftriaxone, as well as cephamycins such as cefoxitin. To a lesser extent, they are also able to hydrolyse penicillins and some monobactams12,47. Chromosomal β-lactamases are produced by many species of Gram-negative bacteria and are especially important in clinical isolates of C. freundii, Enterobacter aerogenes, E. cloacae, S. marcescens and P. aeruginosa because the enzymes can be inducible in these species. The basal expression of chromosomal AmpC-type β-lactamases is generally low, but high expression levels can be induced following exposure to some β-lactams47. AmpC also may be constitutively expressed at high levels in these organisms47. AmpC β-lactamases can also be expressed from plasmids, which have now become widely disseminated. Some of these variants include CMY-1, originating from C. freundii; the MIR and ACT families, originating from Enterobacter spp.; and the DHA family, originating from Morganella morganii47,48,49. Plasmid-encoded AmpC-type enzymes are also called cephamycinases because they readily hydrolyse cefoxitin and other cephamycins. The high level of expression of the plasmid-encoded AmpC variants is mainly due to strong promoters and high gene copy number47. The loss of porin proteins in clinical isolates of K. pneumoniae and Salmonella enterica with plasmid-encoded AmpC enzymes results in resistance to carbapenems50,51,52. Oxacillin-hydrolysing β-lactamases The OXA-type enzymes were traditionally classified as oxacillin-hydrolysing enzymes that are grouped as molecular class D and Bush–Jacoby–Medeiros functional group 2d β-lactamases11. They are now a regarded as a broad group of enzymes that display variability in their substrate profiles and sequences. OXA-type β-lactamases are also distinct from other molecular classes; the amino acid identities between class D and class A or class C β-lactamases are less than 20%53,54. OXA enzymes often have a greater hydrolytic activity against oxacillin than penicillin G, and some members of this group can hydrolyse cephalosporins or carbapenems55. OXA enzymes are only weakly inhibited by clavulanate; however, some of them are inhibited by tazobactam and avibactam56. Several OXA β-lactamases, such as OXA-11 and OXA-14 through OXA-20, are associated with an ESBL phenotype55. OXA-type enzymes have become more important in recent years due to the ability of some members of this class to hydrolyse carbapenems. OXA-type enzymes with a carbapenemase spectrum include OXA-48 and related enzymes such as OXA-162, OXA-163, OXA-181 and OXA-232 that have spread among various species of Enterobacteriaceae and have disseminated throughout Europe and the Mediterranean57. OXA-48-producing strains with increased carbapenem minimal inhibitory concentration (MIC) values often possess a secondary non-enzymatic resistance mechanism58. β-Lactamase inhibitors In Gram-positive bacteria, hydrolysis of the first commercial penicillins by penicillinases was the earliest reported mechanism of resistance to the β-lactams7. Later, the acquisition of PBPs with decreased affinities for common β-lactams became the more important resistance mechanism in Gram-positive cocci59. By contrast, the production of β-lactamases is, and has been, the most common resistance mechanism in Gram-negative bacteria60,61,62. In the mid-1970s, when the plasmid-encoded TEM-1 β-lactamase escaped from enteric bacteria into N. gonorrhoeae and Haemophilus influenzae, pharmaceutical companies initiated extensive research efforts to identify a potent inhibitor of the enzyme63,64. At that time, the key β-lactamases of interest were TEM-1 (previously known as ‘RTEM’), the staphylococcal penicillinases and the chromosomal AmpC-related cephalosporinases. Through the screening of natural products, clavulanic acid (Fig. 1b; Table 2), a natural product isolated from Streptomyces clavuligerus, was identified as a potent time-dependent inhibitor of TEM-1 and the staphylococcal PC1 penicillinase9. Mechanistically, the inhibitor was shown to be a most effective irreversible inactivator of TEM-1 (Fig. 4), with the initial formation of a transiently inhibited intermediate (E–I) resulting in hydrolysed clavulanic acid (I*) and free enzyme (E); a competing pathway involved the formation of an irreversibly inactivated enzyme [E–I]# and fragmented clavulanic acid (I^)65,66. For each molecule of inactivated TEM-1, 115 molecules of clavulanic acid were hydrolysed. Table 2 Comparison of β-lactamase inhibitors Full size table Fig. 4: General reaction mechanism for inhibition of a β-lactamase by a mechanism-based β-lactamase inhibitor. Formation of a reversible complex (E ∙ I, where E is the β-lactamase and I is the β-lactamase inhibitor) may proceed to a stable acyl enzyme (E–I), caused by reaction of the inhibitor with the serine residue in the active site of the enzyme. Three pathways are then possible. Addition of water to the acylated enzyme can result in hydrolysis of the β-lactamase inhibitor (formation of I*) and regeneration of active enzyme (E). Alternatively, the inhibitor could undergo a chemical reaction resulting in fragmentation of the inhibitor, resulting in release of a fragment (I^) into the medium and additional fragmentation products binding to functionally inactive enzyme [E–I]#. Finally, the acyl enzyme may release intact inhibitor (I) and active enzyme (E), or [E + I]. All reactions may be operative at different rates for the same enzyme–inhibitor combination. Accumulation of [E–I]# eventually can lead to complete enzyme inactivation. Full size image Following the discovery of clavulanic acid and the elucidation of the inactivation mechanism, the synthesis of other mechanistically related molecules led to the identification of sulfone-substituted β-lactams as penicillinase inactivators that conformed to a similar inhibitory pathway67,68,69,70. From these studies emerged sulbactam (Fig. 1b; Table 2), a penam sulfone with slightly less potency than clavulanic acid but with modest antibacterial activity against N. gonorrhoeae69 and Acinetobacter spp.71. Later, tazobactam (Fig. 1b; Table 2), a second synthetic penicillanic acid sulfone, was introduced. It has no appreciable antimicrobial activity but greater inhibitory potency than sulbactam and a similar mechanism of inhibition as sulbactam and clavulanic acid68,72 (Table 3). All three inhibitors target the same set of enzymes, primarily class A/group 2 β-lactamases. Clavulanic acid and tazobactam have comparable inhibitory activities against group 2 enzymes, including CTX-M β-lactamases and the ESBL derivatives of TEM-1, TEM-2 and SHV-1, and both have greater potency than sulbactam73. Tazobactam has decreased but measurable activity against class C/group 1 cephalosporinases compared with class A enzymes, whereas sulbactam and clavulanic acid are poor inhibitors of these enzymes69,74. Sulbactam and clavulanic acid are generally less effective against KPC and other serine-based carbapenemases. Inhibition of class D/group 2d enzymes is variable70. No inhibition of the class B/group 3 MBLs has been reported12. Table 3 Mechanisms of action for β-lactamase inhibitors Full size table Tazobactam, sulbactam and clavulanic acid function as ‘suicide’ inactivators, rendering the targeted β-lactamase non-functional (Table 3). Turnover numbers (molecules of inhibitor hydrolysed for each inactivation event) varied for each inhibitor with each enzyme. Sulbactam usually had higher turnover numbers than clavulanic acid or tazobactam for all the β-lactamases tested68,75. With the assistance of X-ray crystallography, covalent adducts with inactivated enzymes have been identified to support the biochemically derived mechanisms (Fig. 4). For example, a structural analysis of tazobactam-inactivated SHV-1 β-lactamase showed that the active-site Ser70 was acylated by tazobactam together with a five-atom tazobactam fragment bound to Ser130 (ref.76). One of the most important considerations of developing a BLI combination is the selection of an accompanying β-lactam that has similar pharmacokinetic properties as the inhibitor. BLI combinations that were introduced into clinical practice in the period of 1984–1993 included1 (Table 2) clavulanic acid paired with amoxicillin as an orally bioavailable agent; clavulanic acid combined with ticarcillin for parenteral delivery of a combination with coverage of Gram-negative infections, including those caused by P. aeruginosa; sulbactam combined with ampicillin, used parenterally for the treatment of urinary tract infections (UTIs) and intra-abdominal infections; and tazobactam combined with piperacillin, with broad-spectrum staphylococcal, Gram-negative and pseudomonal coverage of soft tissue and intra-abdominal and lower respiratory tract infections in hospitalized patients77. The amoxicillin–clavulanic acid combination is currently the most frequently prescribed antimicrobial agent in many paediatric primary care units78. Piperacillin–tazobactam is still frequently used in empiric therapy for nosocomial infections79,80. More recently, tazobactam has been partnered with ceftolozane, and with cefepime in countries outside the United States81,82, providing a broader spectrum of activity of the accompanying cephalosporin but still providing no activity against strains that express serine carbapenemases. Although resistance to the BLI combinations emerged in the 1990s due to mutated forms of the TEM and SHV class A/group 2b β-lactamases, there have been no sustained outbreaks of isolates expressing these enzymes. Several sets of inhibitor-resistant TEM and SHV enzymes have been reported as indicated above. The major cause of resistance to the older BLI combinations has been high levels of β-lactamase activity in the infecting pathogen83. These high levels of activity may be due to increased expression of the targeted enzymes, such as TEM-1 hyperproduction caused by gene amplification in the presence of subinhibitory amounts of tazobactam, or to the presence of multiple β-lactamases produced by a single bacterial species25,50,84. With increasing numbers of ESBLs and carbapenemases that are appearing in multidrug-resistant Gram-negative pathogens, the use of these older combinations is becoming relegated to treating community-acquired respiratory infections with amoxicillin–clavulanic acid or the less serious hospital-acquired infections with ampicillin–sulbactam or piperacillin–tazobactam combinations85. Novel inhibitor classes All three of the early BLIs, clavulanic acid, sulbactam and tazobactam, are effective against many class A β-lactamases but do not provide protection against class B, C and D β-lactamases86 (Table 1). These inhibitors provided excellent coverage for pathogens when ESBLs were first identified; however, they proved to be inefficient against strains that expressed multiple ESBLs, plasmid-encoded AmpC-type β-lactamases or serine carbapenemases such as KPC25,47,86,87. As the KPC β-lactamases and other carbapenemases continue to spread, the need for new inhibitors has arisen86. Fortunately, several new non-β-lactam inhibitor combination agents have been recently approved for clinical use, with many more in the pipeline. Avibactam Avibactam (formerly known as AVE1330A and NXL104) is a bridged diazabicyclo[3.2.1]octanone (DBO) non-β-lactam inhibitor88 (Fig. 1c; Table 2). Compared with clavulanic acid, sulbactam and tazobactam, avibactam provides excellent inhibition of the clinically relevant class A and class C β-lactamases such as CTX-M-15, KPC-2 and AmpC β-lactamases from Enterobacter spp. and P. aeruginosa88,89. In addition, avibactam follows a different mechanism of inhibition than the previous BLIs in that avibactam binds in a covalent, but reversible, manner to most β-lactamases tested, followed by the regeneration of active enzyme and intact inhibitor via deacylation and recyclization of the five-membered urea ring of avibactam (Fig. 4 with the product being [E + I])56,90. In the reaction of avibactam with the KPC-2 enzyme, however, a reaction similar to that observed with clavulanic acid and tazobactam occurs (Fig. 4), in which the (E–I) complex formed during the reaction of KPC-2 with avibactam proceeds to the hydrolysis of avibactam (I*) and regeneration of free enzyme (E)56. Avibactam has been combined with ceftazidime, which provides broad activity against many isolates of Enterobacteriaceae and P. aeruginosa that express one or multiple β-lactamases, including ESBLs, KPC, OXA-48 and AmpC-type enzymes57,89. Although the combination of ceftazidime–avibactam did exhibit improved activity against Bacteroides fragilis, Clostridium perfringens and both Prevotella spp. and Porphyromonas spp. compared with ceftazidime alone, ceftazidime–avibactam is not considered to have sufficient anti-anaerobic activity to provide efficacy against this group of organisms91. A thorough assessment of the pharmacokinetics (PK) and pharmacodynamics (PD) of avibactam determined that the PD driver of efficacy was time dependence rather than concentration dependence, defined as the time that the concentration of avibactam exceeded a critical concentration threshold (%f T > CT). This information was used to determine dose and infusion times for the combination92. Ceftazidime–avibactam completed seven phase III studies and has been approved for the treatment of complicated UTIs (cUTIs), complicated intra-abdominal infections (cIAIs) and hospital-acquired infections. In Europe, the combination is also indicated for the treatment of infections due to aerobic Gram-negative organisms in adult patients with limited treatment options93,94. Avibactam is also in development in combination with aztreonam, which provides activity against Enterobacteriaceae that express both serine β-lactamases and MBLs39,95. The rationale for this combination is that, as noted above, MBLs do not hydrolyse monobactams; however, isolates in which those enzymes occur most often carry one or more ESBLs or serine carbapenemases, thereby requiring the addition of the inhibitor. Aztreonam–avibactam is currently in phase III clinical trials96. Relebactam Relebactam (formerly known as MK-7655) is another DBO that differs from avibactam with the addition of a piperidine ring1 (Fig. 1c; Table 2). It is currently in development as a co-formulated combination with imipenem and the renal dehydropeptidase inhibitor cilastatin97. Relebactam has been combined with imipenem to provide activity against carbapenem-resistant Enterobacteriaceae (CRE) and P. aeruginosa, including strains that produce KPC and class C β-lactamases97,98,99. In addition, imipenem–relebactam showed good activity against isolates of K. pneumoniae and Enterobacter spp. with imipenem resistance caused by impermeability due to the loss of the porin OprD together with the expression of an ESBL or AmpC β-lactamase. By contrast, this combination was not effective in restoring susceptibility to Enterobacteriaceae that express OXA-48 or any of the metallo-carbapenemases98. In addition, relebactam did not restore the activity of imipenem against Acinetobacter baumannii96. In a hollow fibre model, 500 mg of both imipenem and relebactam suppressed growth of a KPC-2-producing K. pneumoniae strain and an OprD-deficient, AmpC-overexpressing isolate of P. aeruginosa after 72 h (ref.99). Phase II clinical trials studying the combination of imipenem–cilastatin–relebactam for the treatment of cUTIs100 and cIAIs101 have been completed. The combination of imipenem–cilastatin–relebactam is currently being studied in a phase III comparative trial for the treatment of hospital-acquired pneumonia and ventilator-associated bacterial pneumonia, with a dose of 250 mg of relebactam combined with 500 mg of imipenem and 500 mg of cilastatin every 6 h compared with piperacillin–tazobactam102. Nacubactam Nacubactam (formerly known as OP0595 and RG6080), a DBO inhibitor that inhibits class A and C serine β-lactamases, is currently being combined with meropenem for development103,104 (Fig. 1c; Table 2). In addition to its β-lactamase inhibitory properties, nacubactam has intrinsic antibacterial activity by targeting PBP2 in isolates of Enterobacteriaceae103,105. When combined with meropenem, nacubactam has shown good activity against a contemporary clinical collection of pathogenic and multidrug-resistant Enterobacteriaceae isolates, including isolates that produce MBLs106. In a mouse model of UTI using simulated human dosing, meropenem–nacubactam demonstrated an antibacterial kill of as much as 6 log10 colony forming units (CFU) compared with a 48 h control when tested against ceftazidime–avibactam-resistant Enterobacteriaceae expressing NDM, KPC, OXA, CTX-M, SHV or TEM β-lactamases107. In a dose fractionation study using the neutropenic mouse thigh model, the percentage of time the free nacubactam concentrations remained above a critical concentration (%f T > a nacubactam threshold CT) is the relevant PK/PD index, as with avibactam108. Nacubactam is currently in phase I clinical studies109. Zidebactam Zidebactam (formerly known as WCK 5107) is another DBO inhibitor with PBP2 binding activity that inhibits class A and C serine β-lactamases and is being partnered with cefepime for further development (Fig. 1c; Table 2). In MIC tests, cefepime–zidebactam was active against Enterobacteriaceae species and P. aeruginosa that express ESBLs, AmpC, KPC and OXA-48 β-lactamases110. Zidebactam is currently in phase I clinical trials111. ETX2514 ETX2514 is a reversible DBO inhibitor that was specifically designed to inhibit class D β-lactamases, in addition to the class A and C enzymes included in the spectrum of activity of the other DBOs112,113 (Fig. 1c; Table 2). Molecular modelling of ETX2514 into the active site of OXA-24 suggested tighter covalent binding than avibactam113. The inhibition of OXA-type β-lactamases allows ETX2514 to be used to target Acinetobacter spp. as OXA β-lactamases are commonly produced in that species. ETX2514 is being combined with sulbactam, which takes advantage of the unique property of sulbactam to bind to essential PBPs in Acinetobacter spp., N. gonorrhoeae and H. influenzae114. As a result, the sulbactam–ETX2514 combination provides good activity against multidrug-resistant A. baumannii113. Sulbactam–ETX2514 has completed a phase II study for safety and efficacy in cUTIs115 and will be developed as a narrow-spectrum agent targeting Acinetobacter spp. Vaborbactam Boronic acid compounds are potent inhibitors of serine proteases116 and, therefore, have been investigated for their potential to be combined with a β-lactam117. Vaborbactam (formerly known as RPX7009), the first boronic acid BLI to be developed, was designed specifically to inhibit KPC carbapenemases but also has activity against other class A and class C β-lactamases117 (Fig. 1d; Table 2). It forms a reversible covalent bond with the targeted β-lactamase, thereby functioning as a competitive inhibitor. Meropenem–vaborbactam has shown good in vitro activity against Klebsiella spp., Enterobacter spp. and Citrobacter spp. that produce KPC enzymes but has less activity in strains that lacked a porin or that overexpressed efflux pumps21,118. By contrast, vaborbactam did not restore activity of meropenem against OXA-48-carbapenemase-producing CRE21. Meropenem–vaborbactam completed phase III trials for UTIs and all infection types against CRE119. It was recently approved with an indication for use in UTIs120. VRNX-5133 VNRX-5133 is a novel boronic acid BLI in clinical development with direct inhibitory activity against class A, C and D serine β-lactamases and VIM and NDM class B MBLs in CRE and P. aeruginosa121 (Fig. 1d; Table 2). The X-ray crystal structure of two clinically relevant β-lactamases, CTX-M-15 and VIM-2, in complex with VNRX-5133 showed that the inhibitor was covalently bound to the catalytic serine residue of CTX-M-15 and that the boron hydroxyl groups interacted with Zn1 and the conserved Asn233 of VIM-2 (ref.122). The cefepime–VNRX-5133 combination has shown both in vitro and in vivo activity against molecularly characterized ESBL-producing and carbapenemase-producing Enterobacteriaceae and P. aeruginosa isolates123,124,125. PK/PD studies showed that the primary pharmacokinetic driver for efficacy for VNRX-5133 was %T > CT (ref.126). VNRX-5133 is currently in phase I clinical trials127. Entmetazobactam Enmetazobactam (formerly AAI101) is a methylated penicillanic acid sulfone BLI related to tazobactam with activity against many serine β-lactamases, particularly ESBLs (Fig. 1b; Table 2). In enzymatic studies, enmetazobactam had low half-maximal inhibitory concentration (IC50) values (≤0.52 µM) when tested with periplasmic extracts of class A β-lactamases128. Enmetazobactam combined with cefepime showed good in vitro activity against an isogenic panel of E. coli strains that express single, clinically relevant β-lactamases and against ESBL-producing clinical isolates of Enterobacteriaceae obtained from US and European medical centres128,129. In the latter study, the cefepime–enmetazobactam combination was at least eight-fold more potent than piperacillin–tazobactam. The in vivo efficacy of simulated human doses of cefepime and cefepime–enmetazobactam in a neutropenic mouse thigh infection model resulted in bacterial reductions of >0.5 log10 CFUs for 12 of 20 carbapenemase-producing E. coli and K. pneumoniae isolates130. Enmetazobactam has completed a phase II study in cUTIs131. ANT431 ANT431, a novel pyridine-2-carboxylic acid that was specifically designed to inhibit MBLs, is currently in preclinical development132 (Fig. 1e; Table 2). ANT431 showed sub-micromolar inhibitory activity against purified NDM-1 and VIM-2 in a biochemical assay, with at least 20-fold weaker inhibition of VIM-1 and IMP-1. When combined with meropenem, ANT431 demonstrated good activity against many clinical isolates in a large panel of Enterobacteriaceae that contain NDM-1, but it demonstrated less potentiation against other MBL-producing variants. It was also efficacious in a mouse thigh model of infection against an E. coli strain expressing NDM-1 (ref.118). Although the investigators indicate that the compound will probably not be developed as a clinical candidate due to its limited range of MBLs that are inhibited, it may function as a prototype for future MBL inhibitors132. Resistance to new combinations Resistance to the new inhibitors or inhibitor combinations has been identified in both the laboratory and in clinical practice. In an early study examining multiple strains of KPC-3-producing E. cloacae and K. pneumoniae, avibactam resistance was selected in vitro both in single-passage and serial-passage experiments133. The majority of mutations associated with resistant strains were caused by amino acid modifications in the Ω-loop of the KPC-3 carbapenemase, particularly the mutation Asp179Tyr that was later identified in clinical isolates resistant to the combination. The double substitution of Asp179Tyr/Thr243Met resulted in even higher MICs in clinical strains134. Interestingly, many of the mutant strains exhibited increased susceptibility to carbapenems and piperacillin–tazobactam133. DBOs such as nacubactam and zidebactam have been shown to bind to PBP2 in enteric bacteria as a secondary mechanism of action. As a result, resistance is not usually associated with β-lactamase-related mechanisms in enteric bacteria105,110. In E. coli, stable mutants with higher nacubactam MICs were selected after exposure to nacubactam. These were caused by activation of the stringent response, thereby compensating for the inactivation of PBP2 by nacubactam105. In P. aeruginosa, upregulation of efflux pumps was associated with elevated cefepime–zidebactam MICs110. Naturally occurring clinical isolates resistant to the ceftazidime–avibactam combination have been reported in E. coli as a result of insertion sequences in PBP3 (refs135,136). Resistance has also been reported during therapeutic use of ceftazidime–avibactam and was shown to be due primarily to mutations in KPC-3, similar to those observed in vitro134. In two of three patients who developed clinical resistance to the combination, meropenem susceptibility was restored. Intrinsic resistance to the meropenem–vaborbactam combination was observed in vitro for OXA-48-producing isolates21. Increased MICs for the combination were also reported for isolates with increased copy numbers of the genes encoding AmpC or KPC, or inactivation of the porin OmpK36, with or without concomitant loss of OmpK35, in K. pneumoniae137. No mutations in KPC enzymes were observed after in vitro selection of resistance with meropenem–vaborbactam in KPC-producing K. pneumoniae isolates137. Results from the meropenem–vaborbactam phase III clinical trial TANGO 1 identified only one pathogen with a meropenem MIC >0.5 μg ml–1, specifically an OXA-48-producing isolate associated with a meropenem MIC of 32 μg ml–1 (ref.119). Conclusions and outlook Hyperproduced AmpC cephalosporinases, ESBLs, serine carbapenemases and MBLs remain the most frequent causes for resistance to β-lactam antibiotics in Gram-negative bacteria. However, addition of the new DBO and boronic acid BLIs to the antibacterial armamentarium has provided the infectious disease community with the ability to counteract many of the organisms that produce serine β-lactamases. Outbreaks of KPC-producing Enterobacteriaceae no longer need to be treated with poorly tolerated, and less efficacious, agents such as the polymyxins when BLI combinations such as ceftazidime–avibactam at this time are working well138. However, it remains to be seen as to how long DBOs or the boronic acid drug combinations will retain their unique properties of providing effective antibacterial activity against multidrug-resistant Gram-negative bacteria. At some point, one can expect that resistance to both classes will become high enough that the combinations will no longer be effective. Most likely, the greatest current challenge remains inhibition of class B MBLs. 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Bradford Contributions K.B. and P.A.B. researched data for the article, made substantial contributions to discussions of the content, wrote the article and reviewed and/or edited the manuscript before submission. Corresponding authors Correspondence to Karen Bush or Patricia A. Bradford. Ethics declarations Competing interests The authors declare competing interests. K.B. serves as an independent consultant for pharmaceutical and biotechnology companies that discover and develop antimicrobial agents. P.A.B. is an independent consultant for companies that work on antibacterial agents. No support or input for the manuscript was provided by any of the companies for which K.B. or P.A.B. consult. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Related links ClinicalTrials.gov: http://www.clinicaltrials.gov Glossary Penicillin-binding proteins (PBPs). Bacterial cell wall synthesizing enzymes that are the killing targets of β-lactam antibiotics. β-Lactamases Bacterial enzymes that hydrolyse β-lactam bonds in β-lactam-containing antibiotics. Integrons Genetic elements, often including resistance determinants, that can be inserted into or excised from bacterial DNA. Porin proteins Bacterial proteins that form channels through the outer membrane of Gram-negative bacteria to allow the entry and exit of small molecules from the periplasmic space of the cell. Hollow fibre model An in vitro pharmacodynamics model that allows bacteria inside a porous membrane to be exposed to varying drug concentrations in a dynamic system that can be programmed to mimic human dosing regimens. Stringent response Refers to the reaction of a bacterial cell to environmental stress. Rights and permissions Reprints and Permissions About this article Cite this article Bush, K., Bradford, P.A. Interplay between β-lactamases and new β-lactamase inhibitors. Nat Rev Microbiol 17, 295–306 (2019). https://doi.org/10.1038/s41579-019-0159-8 Download citation Published 05 March 2019 Issue Date May 2019 DOI https://doi.org/10.1038/s41579-019-0159-8 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Provided by the Springer Nature SharedIt content-sharing initiative Subjects Antibiotics Antimicrobial resistance Bacterial infection Further reading Enzyme-catalyzed biodegradation of penicillin fermentation residues by β-lactamase OtLac from Ochrobactrum tritici Peng Wang Chen Shen Jialin Lu Microbial Cell Factories (2021) High prevalence of blaCTX-M and blaSHV among ESBL producing E. coli isolates from beef cattle in China’s Sichuan-Chongqing Circle Yu-Long Zhang Fang-Yuan Huang Zhi-Cai Zuo Scientific Reports (2021) Detection of carbapenemase producing enterobacteria using an ion sensitive field effect transistor sensor Stathis D. 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