Resistance to Novel β-Lactam–β-Lactamase Inhibitor Combinations

INTRODUCTION

As a class, the β-lactams are the most commonly prescribed and clinically dependable antimicrobials in the United States, representing more than 65% of injected antibiotic prescriptions from 2004 to 2014¹ and 45% of oral antibiotic prescriptions in 2016.² Given their effectiveness, the development of resistance to β-lactam antibiotics creates a major concern for physicians, scientists, and policymakers around the world. This review focuses on the emergence of resistance to the 4 novel β-lactam–β-lactamase inhibitor combinations approved between 2014 and 2019 in the United States: ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam (Fig. 1). The resistance mechanisms that have been reported to date are summarized and the implications of these findings highlighted.

Fig. 1.

Structures of (A) ceftolozane-tazobactam, (B) ceftazidime-avibactam, (C) meropenem-vaborbactam, and (D) imipenem-relebactam. In all panels, the β-lactamase inhibitor (red) is located to the right of the β-lactam partner (blue).

MECHANISM OF ACTION OF β-LACTAM ANTIBIOTICS

Cell wall biosynthesis is critical to bacterial cell division, and most bacteria require a cell wall for survival.³ Cell walls are made of peptidoglycan, long polymers of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) joined in alternating order by β-1,4 glycosidic linkages. Each NAM subunit is attached a short pentapeptide, which is cross-linked between peptidoglycan strands to create a meshlike structure that provides strength to the cell wall. These linkages, which occur between the penultimate D-alanine of 1 peptide and the lysine or diaminopimelic acid of another, are catalyzed by DD-transpeptidases, known as penicillin-binding proteins (PBPs).^4,5

β-Lactam antibiotics enter the transpeptidase active site of PBPs and stereochemically mimic the terminal D-alanine residues of the peptide.⁵ When the active site serine of the PBP attacks the β-lactam ring rather than a peptide bond, it forms a covalent acyl-enzyme complex that deacylates very slowly, crippling the PBP and preventing the final step of cell wall biosynthesis.¹ This leads to potentially endless cycles of futile synthesis and degradation of nonfunctional peptidoglycan, depleting cellular stores of precursors and amplifying cytotoxicity in the process by permitting the entry of water into the cell.⁶

β-LACTAMASES

β-Lactamases are bacterial enzymes that hydrolyze the β-lactam bond of β-lactam antibiotics, rendering them nonfunctional. β-Lactamases are divided into 4 molecular classes by mechanism, conserved residues, and sequence homology. Classes A, C, and D β-lactamases use a conserved serine-based mechanism to hydrolyze the β-lactam bond. Class B metallo-β-lactamases catalyze the hydrolysis of the β-lactam bond using a Zn²⁺-based mechanism.⁷ For purposes of this review, alterations to class A and class C β-lactamases are discussed using the standardized numbering scheme of Ambler and colleagues⁸ and Structural Alignment-based Numbering of class C β-lactamases, respectively.⁹

The basic mechanism used by class A and class C serine β-lactamases involves binding, acylation, and deacylation phases with 2 transition states (Fig. 2). Binding occurs when a substrate associates with the enzyme to form a reversible Michaelis complex. In the acylation phase of a class A or class C serine β-lactamase mechanism, a general base deprotonates the catalytic serine residue, permitting nucleophilic attack of the carbonyl carbon of the β-lactam ring, forming a high-energy transition state, which quickly collapses into the acyl-enzyme complex. Deacylation occurs when a water molecule is deprotonated and nucleophilically attacks the same carbon atom, creating a second high-energy transition state that collapses to restore the serine and release an inactive β-lactam (see Fig. 2).^10–13

Fig. 2.

The class A and class C serine β-lactamase mechanism involves acylation and deacylation through high-energy tetrahedral transition states. In this example with a class C enzyme, B and HB⁺ represent a generic base and conjugate acid, respectively. The identity of the bases may vary by class, enzyme, and substrate.

THE Ω-LOOP OF β-LACTAMASES

Named for its structural resemblance to the Greek letter Ω, the Ω-loop is a highly mobile and dynamic region in β-lactamases¹⁴ and is roughly defined as encompassing residues 164 to 179 in class A enzymes (15 amino acids), residues 188 through 221 (33 amino acids) in class C enzymes, and residues 143 through 173 (30 amino acids) in class D enzymes, although exact designations vary by research group and by family within a class (Fig. 3). The Ω-loop forms the floor of the active site and creates a wall that binds and positions the R1 group of β-lactams, helping to determine substrate specificity.¹⁵ In class A β-lactamases, the Ω-loop is believed to be rigid (rather than flexible) due to hydrogen bonding but remains mobile and able to move as a unit. Simulations suggest class C β-lactamases have a more balanced Ω-loop with both flexible and rigid characteristics and the ability to serve as a mechanical switch, meaning it is able to alternate between more flexible and more rigid states based on changes in the hydrogen bonding network.¹⁴ Amino acid substitutions in the Ω-loop of class A and class C β-lactamases were shown to expand the substrate spectrum of these enzymes toward oxyimino-cephalosporins.^16–19 The precise details and mechanism by which this enhanced ability to hydrolyze these novel cephalosporins with complex R1 side chains occurs still are uncertain.

Fig. 3.

The location of the Ω-loop (blue) in (A) KPC-2 (PDB#: 2OV5), (B) PDC-1 (PDB#: 4GZB), and (C) OXA-48 (PDB#: 4S2P).

β-LACTAMASE INHIBITORS

An established approach to overcoming β-lactam resistance is to reduce the activity of β-lactamases, thus preserving the efficacy of penicillins, cephalosporins, and carbapenem antibiotics. Two approaches to inhibition have targeted β-lactamases successfully, using (1) a suicide, or mechanism-based, inhibitor, and (2) a reversible inhibitor. Suicide inhibitors (including clavulanic acid, sulbactam, and tazobactam) form stable acyl-enzyme complexes, which can undergo postacylation chemistry and fragment or deacylate very slowly. In some cases, these inhibitors permanently inactivate the enzyme in the process. In contrast, reversible inhibitors (including diazabicyclooctanes [DBOs] and boronates) are able to deacylate from the β-lactamase without being modified and proceed to inhibit another β-lactamase molecule.^20–22 Current β-lactamase inhibitors fall into 1 of 3 chemical classes: the suicide inhibitors, which contain β-lactam rings but are less readily hydrolyzed (eg, clavulanic acid, sulbactam and tazobactam); the DBOs, which consist of an 8-membered ring partially analogous to the β-lactam bond (avibactam and relebactam); and the boronic acid transition state inhibitors, which are boronates that mimic transition states (vaborbactam).^20,23

NEWER β-LACTAM AND β-LACTAMASE INHIBITOR COMBINATIONS: CEFTOLOZANE-TAZOBACTAM

Approved by the US Food and Drug Administration (FDA) in 2014, ceftolozane-tazobactam is indicated for use in complicated intra-abdominal infections (cIAIs), complicated urinary tract infections (cUTIs), hospital-acquired bacterial pneumonia (HABP), and ventilator-associated bacterial pneumonia (VABP) in adults.²⁴ Ceftolozane-tazobactam additionally is being or has been investigated for use in adult patients with burns (NCT03002506), indwelling external ventricular drains (NCT03309657), and multidrug-resistant Pseudomonas aeruginosa infections (NCT03510351), and in pediatric patients for gram-negative infections or as perioperative prophylaxis (NCT02266706), for cUTIs (NCT03230838), and for cIAIs (NCT03217136). Ceftolozane-tazobactam is clinically effective against a wide variety of common gram-negative bacteria and some gram positives (Table 1).²⁴

Table 1.

Clinical indications for the use of ceftolozane-tazobactam

Indication	Indicated Bacteria	Patient Population	Clinical Trial
cIAIs	Enterobacter cloacae, Escherichia coli, K oxytoca, K pneumoniae, Proteus mirabilis, P aeruginosa, Bacteroides fragilis, Streptococcus anginosus, Streptococcus constellatus, and Streptococcus salivarius	Adults	ASPECT-cIAI25 NCT01445665 NCT01445678 NCT0273999726
cUTIs, including pyelonephritis	E coli, K pneumoniae, Proteus mirabilis, and P aeruginosa	Adults	ASPECT-cUTI27 NCT01345929 NCT01345955 NCT0272808928
HABP/VABP	Enterobacter cloacae, E coli, Haemophilus influenzae, K oxytoca, K pneumoniae, Proteus mirabilis, P aeruginosa, and S marcescens	Adults	ASPECT-NP29 NCT02070757

Data from Merck & Co., Inc. ZERBAXA (Ceftolozane and Tazobactam) for Injection, for Intravenous Use. Whitehouse Station, NJ 08889 USA; 2014.

Limitations for the combination against indicated organisms include P aeruginosa or Enterobacterales that carry class A and class B carbapenemases (eg, KPC, VIM, NDM, and IMP) or class A, class C, and class D extended-spectrum β-lactamases (ESBLs) (eg, GES-6, PER-1, FOX-4, and OXA-539) that are not readily inhibited by tazobactam.^30–34 E coli–producing class A ESBLs are more susceptible to ceftolozane-tazobactam than K pneumoniae and Enterobacter cloacae–expressing class A ESBLs.^35–38 Chromosomal and acquired bla_AmpCs likely contribute to the former phenotype, because the hyperproduction of AmpCs or class A ESBLs was shown to reduce efficacy of ceftolozane-tazobactam.^{33,36,39–41}

Ceftolozane is an expanded-spectrum cephalosporin that was developed with the intention of creating a novel, antipseudomonal β-lactam antibiotic that targets PBP3.⁴² Ceftolozane is modeled on the success of the closely related cephalosporin, ceftazidime, which is a first-line treatment of P aeruginosa infections. Specifically, ceftolozane was designed to be stable to the presence of Pseudomonas-derived cephalosporinase (PDC),⁴³ the class C or AmpC β-lactamase of P aeruginosa.⁴⁴ Unfortunately, as with other oxyimino-cephalosporins, ceftolozane is susceptible to hydrolysis by certain ESBLs (eg, PER-1) and carbapenemases (eg, KPCs) that often occur in conjunction with other ceftolozane-susceptible enzymes and overexpression of class C enzymes reduces its potency.⁴⁵ Moreover, ceftolozane is readily hydrolyzed by class B metallo-β-lactamases (eg, VIM, IMP, and NDM) and activity against bacteria producing class D OXA β-lactamases is variable.⁴⁶

Tazobactam is a penicillin-based sulfone derivative developed as a β-lactamase inhibitor⁴⁷ that inactivates most class A β-lactamases. Tazobactam demonstrates variable activity against bacteria producing class A carbapenemases, class C β-lactamases, and class D β-lactamases.^48,49 By using tazobactam in this combination, the goal was to inhibit class A ESBLs (eg, CTX-M) and tazobactam-susceptible class C β-lactamases (eg, CMY), thus extending the usefulness of the combination.^33,35,45

REPORTS AND MECHANISMS OF RESISTANCE: CEFTOLOZANE-TAZOBACTAM

In Table 2, the reports of resistance to ceftolozane-tazobactam available to date are summarized. The most frequently reported cause of ceftolozane-tazobactam resistance in isolates for which it is indicated is alterations in PDC, the chromosomally encoded class C β-lactamase of P aeruginosa. Amino acid substitutions, insertions, and deletions in PDC were found in broad survey studies, individual case reports, and laboratory selection experiments, suggesting they can emerge in a variety of ways. Additional mechanisms including acquisition of rare class A β-lactamases as well as amino acid substitutions in OXA enzymes are described herein (see also above).

Table 2.

Mechanisms of resistance to ceftolozane-tazobactam

Type	Organism	Mechanism(s)	Relevant Amino Acid Substitutions in β-Lactamase	Minimum Inhibitory Concentration of Isolatea	Minimum Inhibitory Concentration in a Susceptible Backgroundb	Reference
Case report	P aeruginosa	PDC	G156D	≥64	≥64	57
Case report	P aeruginosa	Overexpression of PDC, loss of oprD	T96I	>32	32	52
Case report	P aeruginosa	Overexpression of PDC, loss of oprD	E219K	>32	16	52
Case report	P aeruginosa	Overexpression of PDC, loss of oprD	ΔG202-E219	32	32	52
Case report	P aeruginosa	Overexpression of PDC, loss of oprD, mutations in mexD, mexT, mexI and mexR	R52Q, T79 A, G156D	≥256	N/A	61
Case report	P aeruginosa	Overexpression of PDC	V211A	64	N/A	50
Case report	P aeruginosa	PDC, mutation in mexR	ΔP208-G214	>128	N/A	50
Case report	P aeruginosa	PDC, loss of oprD	G156D	32–48	N/A	60
Laboratory strain	P aeruginosa	Overexpression of PDC	G156D	64	32	55
Laboratory strain	P aeruginosa	Overexpression of PDC	E219K, V329I	32	64	55
Laboratory strain	P aeruginosa	Overexpression of PDC	F121L, Q130R, E219K, V329I	128	128	55
Survey	P aeruginosa	Overexpression of PDC, loss of oprD	Q128R, V211T, S279T	256	N/A	54
Survey	P aeruginosa	PDC, loss of oprD and mutations in mexT	A5V, V211A, G214R, G220S	12	N/A	54
Survey	P aeruginosa	Overexpression of PDC, loss of oprD	Q128R, V211A, G220S	256	N/A	54
Survey	P aeruginosa	Overexpression of PDC, loss of oprD	V211A	256	N/A	54
Survey	P aeruginosa	PDC	P153L	N/A	8	51
Survey	P aeruginosa	PDC	G214R	N/A	8	51
Survey	P aeruginosa	PDC	N346I	N/A	4–8	51
Survey	P aeruginosa	PDC	R100H, G214R	N/A	16	51
Survey	P aeruginosa	PDC	E219K	N/A	64 to >64	51
Survey	P aeruginosa	PDC	E219G	N/A	32	51
Survey	P aeruginosa	PDC	F121L, M174L	N/A	16	51
Survey	P aeruginosa	PDC	V211A, N346I	N/A	16	51
Survey	P aeruginosa	PDC	ΔT289-M291	N/A	4–8	51
Survey	P aeruginosa	PDC	ΔT289-A292	N/A	16	51
Case report	P aeruginosa	Overexpression of PDC, loss of oprD	ΔG202-E219	32	N/A	56
Survey	P aeruginosa	Overexpression of PDC and/or MexAB-OprM or MexXY-OprM and/or loss of oprD		8–16	N/A	68
Case report	P aeruginosa	PAC-1		>128	>128	74
Case report	P aeruginosa	FOX-4		16	16	34
Laboratory strain	P aeruginosa	GES-1		N/A	32	34
Laboratory strain	P aeruginosa	GES-2	G170N	N/A	16	34
Laboratory strain	P aeruginosa	GES-5	G170S	N/A	8	34
Laboratory Strain	P aeruginosa	GES-6	E104K, G170S	N/A	32	34
Case report	P aeruginosa	GES-6	E104K, G170S	32	64	75
Laboratory strain	P aeruginosa	PER-1		N/A	512	34
Laboratory strain	P aeruginosa	BEL-1		N/A	8	34
Laboratory strain	P aeruginosa	BEL-2		N/A	32	34
Survey	P aeruginosa	BEL-3	P160S	16	N/A	80
Laboratory strain	P aeruginosa	VEB-1		N/A	64	34
Case report	P aeruginosa	OXA-2, loss of oprD	Duplication D149	>32	16	78
Case report	P aeruginosa	OXA-2, VIM-20, mutation of mexZ mexX, and mexB, loss of oprD	ΔI159 + E160 K in OXA-2	>32	32	79
Survey	P aeruginosa	OXA-2	W159R	16–32	16	68,80
Survey	P aeruginosa	OXA-10	N73S	64	N/A	80
Survey	P aeruginosa	OXA-101		32	N/A	80
Case report	P aeruginosa	OXA-10, loss of oprD	N146S	32	N/A	52
Laboratory strain	E coli	GES-1		N/A	8	31
Laboratory strain	E coli	GES-5	G170S	N/A	8	31
Laboratory strain	E coli	GES-6	E104K, G170S	N/A	64	31
Case report	E coli	GES-19, GES-26		N/A	48	77
Laboratory strain	E coli	PER-1		N/A	128	31
Laboratory strain	E coli	BEL-1		N/A	4	31
Laboratory strain	E coli	BEL-2		N/A	8	31

Tazobactam is maintained at 4 μg/mL when in combination with ceftolozane.⁸¹

P aeruginosa breakpoints: intermediate = 8 μg/mL; resistant ≥16 μg/mL.

Enterobacterales breakpoints: intermediate = 4 μg/mL; resistant ≥8 μg/mL.

Abbreviation: NA, not available.

^aThe MIC values for the isolate represent the MIC values obtained for either a clinical isolate obtained from a patient in a case study or part of a surveillance study or a laboratory-selected strain.

^bThe MIC values in a susceptible background represent the MIC value after the bla gene for the β-lactamase of interest was cloned and expressed in a susceptible strain.

Data from Clinical and Laboratory Standards Institute (CLSI). M100: Performance Standards for Antimicrobial Susceptibility Testing. 30th ed.; 2020.

Pseudomonas-Derived Cephalosporinase Variants that Confer Ceftolozane-Tazobactam Resistance

Not surprisingly, given the role it plays in β-lactamase function, the Ω-loop of PDC appears to be an important region for amino acid substitutions leading to ceftolozane-tazobactam resistance when these β-lactamases are expressed in bacteria, with V211A,^50,51 G214R,⁵¹ E219G,⁵¹ E219K,^51,52 and Y221H⁵¹ leading to varying levels of resistance (minimum inhibitory concentrations [MICs] range for clinical isolates: 32–>256 μg/mL) even as single amino acid substitutions (see Fig. 3, Table 2).⁵³ Several of these substitutions also occur in tandem with others, including Q128R V211T S279T,⁵⁴ A5V V211A G214R G220S,⁵⁴ Q128R V211A G220S,⁵⁴ E219K V329I,⁵⁵ F121L Q130R E219K V329I,⁵⁵ R100H G214R,⁵¹ and V211A N346I⁵¹ (see Fig. 3). Of these Ω-loop substitutions, the V211A, E219K, and E219G variants of PDC surfaced alone and E219K in conjunction with G154R substitution during treatment of patients.^50,52,56

Outside the Ω-loop, substitutions are found in several regions of the enzyme but do not seem to cluster in a specific area. These substitutions include T96I,^57,58 F121L,^56,59 G156D alone^55,57,60 and in combination with R52Q and T79A,⁶¹ P154L,⁵¹ L293P, N346I,⁵¹ and F121L M174L⁵¹ (Fig. 4). At this time, it is unknown if these substitutions outside the Ω loop serve to stabilize the protein (serve as a global suppressor) or specifically enhance catalytic activity. The T96I, F121L, and G156D variants of PDC emerged during treatment of patients for infections caused by P aeruginosa and ceftolozane-tazobactam MICs were elevated from 1 μg/mL to 4 μg/mL at the start of treatment to 32 μg/mL to 64 μg/mL during treatment.^{52,56,57,60,61} Moreover, multiple amino acid deletions leading to ceftolozane-tazobactam resistance were reported in PDC and are found both in the Ω-loop, deleting residues P208-G214,^50,58,61 G202-E219,^53,56 and G204-Y221⁵⁸ as well as the R2 loop, deleting residues T289-P290, T289-M291, T289-A292, and L293-Q294.⁵¹ The R2 loop deletions tend to be associated with relatively low-level resistance when expressed in P aeruginosa 4098 (MIC range: 4–16 μg/mL)⁵¹ (see Fig. 4, Table 2). Of these loop deletions, to date, only Ω-loop deletion variants of PDC in P aeruginosa were reported to have surfaced in the clinic during treatment and resulted in ceftolozane-tazobactam MICs of 32 μg/mL to 256 μg/mL.^50,52,56,58

Fig. 4.

PDC crystal structure highlighting the major active site motifs (blue: Ω-loop; magenta: S₆₄X₆₅X₆₆K₆₇ motif; dark green: K₃₁₅T₃₁₆G₃₁₆ motif; light green: Y₁₅₀X₁₅₁K₁₅₂ motif; and pink: R2 loop) (left) and the location of the amino acid substitutions, insertions, and deletions (red: deletion; yellow: substitution; and orange: deletion or substitution) that confer ceftolozane-tazobactam resistance (right).

The Molecular Basis of the Resistance Phenotype: PDC E219K, an Ω-Loop Variant

Among the best-characterized β-lactamase variants conferring ceftolozane-tazobactam resistance is the E219K variant of PDC. When the negatively charged glutamic acid (E) residue at 219 is changed to a positively charged lysine (K) and expressed in P aeruginosa PAO1 Δbla_PDC, the ceftolozane-tazobactam MIC increases from 0.5 μg/mL to 16 μg/mL.⁵² Steady-state kinetic characterization of the purified PDC-3 E219K variant with ceftolozane revealed a K_m of 341 μM ± 64 μM and a k_cat of 10 ± 1 s⁻¹ compared with the wild-type PDC-3 enzyme, which had undetectable hydrolysis of ceftolozane and interacted with ceftolozane very poorly with a K_{i app} of 1300 μM.⁶² Moreover, electrospray-ionization mass spectrometry used to capture β-lactamase–ceftolozane adducts supported the kinetic observations, because, true to its design, ceftolozane was not detected bound to the wild-type Pseudomonas AmpC (PDC-3); conversely, ceftolozane was hydrolyzed by the PDC-3 E219K variant and acyl-enzyme complexes were not detected.⁶² Thermal stability assays revealed that the PDC-3 E219K variant possessed a lower T_m of 45°C compared with 52°C for PDC-3; these data suggest that the PDC-3 E219K variant is less stable.

The molecular mechanism that allows the PDC-3 E219K variant to hydrolyze ceftolozane is perhaps best revealed by using classic atomistic molecular dynamics and well-tempered metadynamic simulations that model the interactions between the enzyme and substrate. The metadynamic simulations uncovered that the PDC-3 E219K variant was more conformationally flexible than the wild-type PDC-3. Moreover, the molecular dynamics showed that the flexibility of the PDC-3 E219K variant allows the nearby Y221 residue to rotate perpendicular to its usual position and open a hidden cavity adjacent to the active site (Fig. 5). This cavity is better able to accommodate the R1 group of ceftolozane (which normally faces a steric clash with Y221), allowing for better positioning of ceftolozane within the active site of the PDC-3 E219K variant to facilitate hydrolysis.

Fig. 5.

Ceftolozane (pink) docked in the active site of the PDC-3 E219K variant. Hydrogen bonds between ceftolozane and residues are indicated with green dashed lines, the catalytic S64 is in cyan, and the Ω-loop in blue. Two conformations of Y221 showcase the increased flexibility of the variant: purple represents a conformation found in both PDC-3 and the PDC-3 E219K variant whereas green represents a conformation found only in the PDC-3 E219K variant, the E219K substitution enables the movement between these conformations, allowing ceftolozane to enter the active site and bind while maintaining residues in catalytically favorable conformations.

Other Contributing Resistance Factors in Pseudomonas aeruginosa

Variants of PDC that result in ceftolozane-tazobactam resistance are found in strains that also have decreased expression of oprD and/or up-regulation of mexAB-oprM or mexXY-oprM efflux pumps and/or de-repression of bla_PDC expression, which is normally expressed at a low basal level and induced by the presence of β-lactam antibiotics( Fig. 6, see Table 2).^63–67 Regarding ceftolozane-tazobactam resistance, OprD does not appear to be necessary for entry of either compound and neither component is greatly impacted by hyperexpression of efflux pumps.^43,68 Controlled (eg, ΔampD and ΔdacB) de-repression of wild-type bla_PDC-1 in a P aeruginosa PAO1 background revealed that ceftolozane MICs are not impacted by hyperexpression of bla_PDC-1.⁶⁹ In conjunction with other factors (eg, bla_PDC mutants and other bla genes), however, de-repression of bla_PDC (eg, mutations in ampR, dacB, ampG, and ampD) was shown to elevate ceftolozane-tazobactam MICs.^30,52,55 Importantly, high-level resistance due to overexpression of bla_PDC variants was associated with the hypermutator background of P aeruginosa.⁵⁵ Other potential contributors toward ceftolozane-tazobactam resistance in P aeruginosa are single amino acid substitutions in PBP3 (R504 C and F533 L); this may be an emerging phenotype due to selective pressure.^30,59

Fig. 6.

Regulation of bla_PDC in P aeruginosa (61–64). (A) During normal cell growth, bacteria degrade approximately half their peptidoglycan and recycle approximately 90% of these degradation products or Glc-NAc-1,6-anhydro-MurNAc-peptides (green rectangular lollipops), which are transported into the cytoplasm via AmpG. In the cytosol, NagZ catalyzes the formation of 1,6-anhydro-MurNAc-peptides (green circular lollipops) that are activating peptides of AmpR, a LysR-type transcriptional regulator that controls expression of bla_ampC. AmpD, an N-acetylmuramyl-L-alanine amidase, cleaves the peptide (green stick) from the 1,6-anhydro-MurNAc (green circle) and the components enter the recycling pathway, keeping the cytoplasmic levels of activating 1,6-anhydro-MurNAc-peptides low and producing UDP-MurNAc-pentapeptides (green pentagonal lollipops) that are suppressing peptides that bind AmpR to repress the transcription of bla_PDC. (B) In the presence of β-lactam antibiotics, the low molecular mass PBP4 (DacB) is inhibited (along with other PBPs), leading to an increase and shift in the composition of the Glc-NAc-1,6-anhydro-MurNAc-peptides entering the cytoplasm. This increase ultimately overpowers the capacity of AmpD to cleave the peptide from 1,6-anhydro-MurNAc, leading to a buildup of 1,6-anhydro-MurNAc-peptide in the cell. The 1,6-anhydro-MurNAc-peptide is then able to bind AmpR, activating transcription of bla_ampC. The AmpC β-lactamases are exported to the periplasm where they inactivate β-lactams. (C) Mutations in ampD are the most common cause of derepressed bla_ampC by severely crippling the production and/or activity of AmpD, levels of 1,6-anhydro-MurNAc-peptides greatly increase within the cell, bind to AmpR, and induce the production of high levels of bla_ampC.

The Impact of Other AmpCs

Resistance to ceftolozane-tazobactam in class C β-lactamases other than PDC is reported much less frequently or studied, but the Y221H substitution in CMY-2 was also shown to result in an elevated ceftolozane-tazobactam MIC (2.5 μg/mL).⁷⁰ Other Ω-loop substitutions leading to ceftolozane-tazobactam resistance in P aeruginosa have been reported in other species producing class C β-lactamases, but in the context of nonsusceptibility to different substrates (eg, ceftazidime), including E219K in Citrobacter freundii AmpC⁷¹ and V211A combined with L239S in CMY-95.⁷²

Additionally, SRT-1 of Serratia marcescens has lysine in the 219 position and exhibits better hydrolysis of several cephalosporins (eg, ceftazidime) than the closely related SST-1 with glutamic acid at 219.⁷³ Additionally, 4 cases of P aeruginosa harboring bla_PAC-1 were reported in patients repatriated from Mauritius and Afghanistan.⁷⁴ PAC-1 is a unique class C β-lactamase with 47% sequence identity to PDC-1 and confers ceftolozane-tazobactam resistance; the introduction of bla_PAC-1 into P aeruginosa PAO1 increased the ceftolozane-tazobactam MIC from less than or equal to 0.5 μg/mL to greater than 128 μg/mL. Recently, the FOX-4 cephamycinase was found responsible for elevated ceftolozane-tazobactam MICs (16 μg/mL) in a P aeruginosa clinical isolate.³⁴ Much remains to be explored with non-PDC AmpCs and their involvement in ceftolozane-tazobactam resistance.

Uncommon Class A β-Lactamases

The acquisition of several different class A β-lactamases (eg, GES, PER-1, BEL-1, BEL-2, and VEB-1) has been associated with the emergence of ceftolozane-tazobactam resistance.^{31,36,53,59,75,76} Depending on the strain background in which these β-lactamases are produced, P aeruginosa versus E coli, the ceftolozane-tazobactam MICs vary (eg, P aeruginosa with bla_GES-1 MIC = 32 μg/mL vs E coli with bla_GES-1 MIC = 8 μg/mL)³¹ (see Table 4). When the combination of bla_GES-19 and bla_GES-26 was expressed in E coli TG1, the MIC values for ceftolozane-tazobactam increased to 48 μg/mL compared with an MIC of greater than 256 μg/mL when in P aeruginosa.⁷⁷ The production of PER-1 in P aeruginosa PA01 resulted in high level ceftolozane-tazobactam resistance (MIC: 512 μg/mL).³¹ Except for GES β-lactamases, rare class A carbapenemases (eg, IMI and SME) are poor cephalosporinases; thus, when expressed, these strains usually are susceptible to ceftolozane-tazobactam. ³⁶

Table 4.

Mechanisms of resistance to ceftazidime-avibactam

Type	Organism	Mechanism(s)	Relevant Amino Acid Substitutions in β-Lactamase	Minimum Inhibitory Concentration of Isolatea	Minimum Inhibitory Concentration in a Susceptible Backgroundb	Reference
Laboratory	E coli	KPC-2	R164A	N/A	16	102
Laboratory	E coli	KPC-2	R164P	N/A	64	102
Laboratory	E coli	KPC-2	D179A	N/A	64	102
Laboratory	E coli	KPC-2	D179Q	N/A	32	102
Laboratory	E coli	KPC-2	D179N	N/A	32	102
Laboratory	E coli	KPC-2	D179Y	N/A	32	106
Survey	K pneumoniae	KPC-2, SHV-12, TEM-1, and loss of ompK35, ompk36, and ompK37		16	N/A	166
Survey	E coli	KPC-2, TEM-1, 344_ins_TIPY_345_PBP3		8	N/A	153
Case	K pneumoniae	Overexpression of KPC-2, loss of ompK35 and ompK36		12	N/A	108
Laboratory	C freundii	KPC-2, overexpression of acrA, loss of ompF	D176Y	32	8	110
Laboratory	C freundii	KPC-2, overexpression of acrA, loss of ompF	R146S, P147L	64	8	110
Laboratory	C freundii	KPC-2, overexpression of acrA, loss of ompF	D179Y	64	8	110
Case	K pneumoniae	KPC-2, SHV-11, SHV-12, TEM-1, loss of ompK35, ompK36, and ompK37	D179Y	>256		120
Survey	K pneumoniae	KPC-2	E166_ins_EL_L167, V278_ins_ SEAV_A281	128	64	117
Case	K pneumoniae	KPC-2	L169P	16	4	125
Case	K pneumoniae	KPC-2, SHV-11	L259_ins_AVYTRAPNKDDKHSE_V260	>16		127
Laboratory	E coli	KPC-2	ΔG242-T243	N/A	24	126
Laboratory	K pneumoniae	KPC-3	S181_ins_S_S182	64		103
Laboratory	K pneumoniae	KPC-3	D179Y	8–64		103
Laboratory	K pneumoniae	KPC-3	D163G	32		103
Laboratory	Enterobacter cloacae	KPC-3	S181_ins_SS_S182	32		103
Laboratory	Enterobacter cloacae	KPC-3	P174L	8–16		103
Laboratory	Enterobacter cloacae	KPC-3	T243P	32		103
Laboratory	Enterobacter cloacae	KPC-3	T265_ins_AR_R266	16		103
Laboratory	Enterobacter cloacae	KPC-3	P183_ins-RAVTTSSP_R184	128		103
Case	K pneumoniae	KPC-3, SHV-11, TEM-1, OXA-9 and loss of ompk36	D179Y	64–256	8	104
Case	K pneumoniae	KPC-3, SHV-11, TEM-1, OXA-9 and loss of ompk36	D179Y, T243M	256	64	104
Case	K pneumoniae	KPC-3, SHV-11, TEM-1, OXA-9	V240G	32	2	104
Case	K pneumoniae	KPC-3, SHV-11, TEM-1, OXA-9 and loss of ompk35	A177E, D179Y	128	N/A	107
Case	K pneumoniae	Overexpression of KPC-3, loss of ompk35 and ompk35, SHV-11, SHV-12		32		145
Survey	K pneumoniae	KPC-3	ΔE166-L167	16–32	8–16	117
Survey	K pneumoniae	KPC-3	L7P, D179Y, T243M	256	N/A	117
Case	K pneumoniae	KPC-3, SHV-11, TEM-1A, OXA-9 loss of ompK35 and ompK36	V240A	16	N/A	118
Laboratory	E coli	KPC-3	ΔG242-T243	N/A	12	126
Laboratory	K pneumoniae	KPC-3	D163G	32	N/A	109
Laboratory	K pneumoniae	KPC-3	R164S	64	N/A	109
Laboratory	K pneumoniae	KPC-3	E168_ins_EL_L169	32	N/A	109
Laboratory	K pneumoniae	KPC-3	L169_ins-KL_N170	64	N/A	109
Laboratory	K pneumoniae	KPC-3	N170D	32	N/A	109
Laboratory	K pneumoniae	KPC-3	N170D + ΔS171	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	A172S	64	N/A	109
Laboratory	K pneumoniae	KPC-3	A172T + T243A	64	N/A	109
Laboratory	K pneumoniae	KPC-3	A172P	64	N/A	109
Laboratory	K pneumoniae	KPC-3	P174L	64	N/A	109
Laboratory	K pneumoniae	KPC-3	P174_ins_PGDARD_D179	64	N/A	109
Laboratory	K pneumoniae	KPC-3	G175V	64	N/A	109
Laboratory	K pneumoniae	KPC-3	D176Y	32	N/A	109
Laboratory	K pneumoniae	KPC-3	D179A	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	D179H	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	D179N	32	N/A	109
Laboratory	K pneumoniae	KPC-3	D179Y	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	ΔV240	128	N/A	109
Laboratory	K pneumoniae	KPC-3	Y241N	64	N/A	109
Laboratory	K pneumoniae	KPC-3	T243M	32	N/A	109
Laboratory	K pneumoniae	KPC-3	Y263_ins_YTRAPN_N269	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	Y263_ins_YTRAPNKDDKYSEAV_V278	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	R266_ins_RAS_P268	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	R266_ins_RAPNKDDKYS_S275	>256	N/A	109
Laboratory	K pneumoniae	KPC-3	P268_ins_PN_N269	64	N/A	109
Laboratory	K pneumoniae	KPC-3	K270_ins_ KD_D271	128	N/A	109
Case	K pneumoniae	KPC-3	K270_PNK_D271	>128	128	128
Case	K pneumoniae	KPC-3	L169P, A172T	>16		122
Case	K pneumoniae	KPC-3	A172T, T243A	>16		122
Case	K pneumoniae	KPC-3	A172T	>16		122
Survey	K oxytoca	SHV-12, TEM-1		16		134
Laboratory	E coli	CTX-M-15	S130G, L169Q	N/A	16	133
Case	K pneumoniae	CTX-M-14, OXA-48	CTX-M-14: P167S, T264I	32		132
Laboratory	E coli	GES-5		N/A	0.5	31
Laboratory	E coli	PER-1		N/A	8	31
Laboratory	E coli	GES-19, GES-26		N/A	256	77
Case	K pneumoniae	VEB-1, TEM-1, 0XA-10	K234R	32–128		138
Survey	E coli	CMY-42, CTX-M-15, OXA-1, 333_ins_YRIK_334_PBP3		8	N/A	154
Survey	E coli	CMY-42, CMY-2, OXA-1, OXA-9, TEM-1, 333_ins_YRIK_334_PBP3		8	N/A	154
Survey	K pneumoniae	DHA-1, loss of ompK35 and ompK36		16	N/A	41
Survey	Enterobacter cloacae	AmpC	ΔS289-A294	64	N/A	140
Laboratory	Enterobacter cloacae	AmpC	G156R	64	N/A	139
Laboratory	Enterobacter cloacae	AmpC	G156D	16	N/A	139
Laboratory	C freundii	AmpC	N346Y	16	N/A	139
Laboratory	C freundii	AmpC	R148P	16	N/A	139
Laboratory	C freundii	AmpC	R148H	32	N/A	139
Survey	K pneumoniae	OXA-48, SHV		16	N/A	161
Survey	S marcescens	OXA-48, CTX-M-22, SHV		64	N/A	161
Survey	P aeruginosa	GES-5		16	N/A	31
Case	P aeruginosa	GES-19, GES-26		128 to >256	N/A	77
Survey	P aeruginosa	PER-1		64	N/A	31
Laboratory	P aeruginosa	PDC	ΔR210-E219	64–256	N/A	141
Laboratory	P aeruginosa	PDC	ΔK204a-G222	64	N/A	141
Laboratory	P aeruginosa	PDC	ΔD217-Y221	256	N/A	141
Laboratory/Case	P aeruginosa	PDC	G156D	128	>32	57,60,141,142
Case	P aeruginosa	Overexpression of PDC, loss of oprD	T96I	16–32	8	52
Case	P aeruginosa	Overexpression of PDC, loss of oprD	E219K	>32	16	52
Case	P aeruginosa	Overexpression of PDC, loss of oprD	ΔG202-E219	32	16	52
Survey	P aeruginosa	Overexpression of PDC and MexAB-OprM, loss of oprD		512	N/A	52
Case	P aeruginosa	Overexpression of PDC	V211A	>64	N/A	50
Case	P aeruginosa	PDC, mutation in mexR	ΔP208-G214	>64	N/A	50
Survey	P aeruginosa	Overexpression of PDC, loss of oprD	Q128R, V211T, S279T	256	N/A	54
Survey	P aeruginosa	Overexpression of PDC, loss of oprD	Q128R, V211A, G220S	256	N/A	54
Survey	P aeruginosa	Overexpression of PDC, loss of oprD	V211A	256	N/A	54
Case	P aeruginosa	PAC-1		>128	>128	74
Case	P aeruginosa	FOX-4		32	32	34
Case	P aeruginosa	OXA-2, loss of oprD	Duplication D149	>32	32	78
Case	P aeruginosa	OXA-10, loss of oprD	N146S	32	N/A	52
Case	P aeruginosa	OXA-2, VIM-20, mutation of mexZ mexX, and mexB, loss of oprD	ΔI159 + E160K in OXA-2	32	32	79

Avibactam is maintained at 4 μg/mL when in combination with ceftazidime.⁸¹

P aeruginosa breakpoint: resistant greater than 8 μg/mL.

Enterobacterales breakpoint: resistant greater than 8 μg/mL.

Abbreviation: NA, not available.

^aThe MIC values for the isolate represent the MIC values obtained for either a clinical isolate obtained from a patient in a case study or part of a surveillance study or a laboratory-selected strain.

^bThe MIC values in a clean background represent the MIC value after the bla gene for the β-lactamase of interest was cloned and expressed in a susceptible strain.

Data from Clinical and Laboratory Standards Institute (CLSI). M100: Performance Standards for Antimicrobial Susceptibility Testing. 30th ed.; 2020.

Resistance Observed in Class D β-Lactamases

Ceftolozane-tazobactam–resistant OXA variants (eg, OXA-14) also were identified in surveillance studies and have emerged during treatment of infections due to P aeruginosa^{52,56,68,78–80} (see Table 2). Many of these OXA variants acquired a single amino acid substitution, such as a strain of P aeruginosa producing the OXA-10 N146S variant possessed a ceftolozane-tazobactam MIC of 64 μg/mL.⁵² The continued evolution of narrow-spectrum oxacillinases in P aeruginosa (such as OXA-14) to ceftolozane-tazobactam resistant variants may represent an emerging challenge when using ceftolozane-tazobactam.

NEWER β-LACTAM AND β-LACTAMASE INHIBITOR COMBINATIONS: CEFTAZIDIME-AVIBACTAM

Ceftazidime-avibactam was approved by the FDA in 2015 for adult and pediatric use in cIAIs (with metronidazole) and cUTIs, including pyelonephritis, and for adult use in HABP and VABP.⁸² Ceftazidime-avibactam is being actively investigated for use in adult cystic fibrosis patients (NCT02504827), for nosocomial pneumonia in pediatric patients (NCT04040621), and in neonates and infants with gram-negative infections (NCT04126031).

Ceftazidime-avibactam is effective against a variety of gram-negative bacteria (Table 3) (Ceftazidime-Avibactam PI). Limitations for the combination against indicated organisms include P aeruginosa or Enterobacterales that carry class B carbapenemases (eg, VIM, NDM, and IMP)^83,84 and non–OXA-48–like class D β-lactamases with ESBL activity (eg, OXA-2 variants).^78,79,85,86

Table 3.

Clinical indications for the use of ceftazidime-avibactam

Indication	Indicated Bacteria	Patient Population
cIAI (with metronidazole)	E coli, K pneumoniae, Proteus mirabilis, Enterobacter cloacae, K oxytoca, C freundii complex, and P aeruginosa	Adult and pediatric (≥3 mo)	RECLAIM 1 and 287 NCT01499290 NCT01500239 REPRISE88 NCT01644643 NCT0247573389
cUTI, including pyelonephritis	E coli, K pneumoniae, Enterobacter cloacae, C freundii complex, Proteus mirabilis, and P aeruginosa	Adult and pediatric (≥3 mo)	RECAPTURE90 NCT01595438 NCT01599806 REPRISE88 NCT01644643 NCT02497781
HABP/VABP	K pneumoniae, Enterobacter cloacae, E coli, S marcescens, Proteus mirabilis, P aeruginosa, and Haemophilus influenzae	Adult	REPROVE91 NCT0180809292

Data from Allergan USA, Inc. AVYCAZ (Ceftazidime and Avibactam) for Injection, for Intravenous Use. Madison, NJ 07940 USA.; 2019.

Why is this combination so important? Ceftazidime is a broad-spectrum amino-thiazolyl cephalosporin originally approved by the FDA in July 1985. Ceftazidime demonstrates potent activity against a wide variety of gram-negative bacteria and some gram-positive bacteria, with particular strengths against P aeruginosa and Enterobacterales, including strains expressing many important β-lactamases.^93,94 Unfortunately, resistance to ceftazidime rapidly emerged (presence of ESBLs in many enteric bacilli, such as E coli and K pneumoniae and the overexpression of class C β-lactamases among other mechanisms) and the drug became less attractive and use was heavily monitored.^20,95,96 Ceftazidime-avibactam helped fill an important gap in the spectrum of other β-lactamases known and evolving at the time with its potent activity against P aeruginosa and ESBLs. In fact, an early article went so far as to describe it as “the most effective antibiotic thus far known against P. aeruginosa.”⁹⁴

Avibactam is the first of a novel class of β-lactamase inhibitors known as the DBOs and has a wide spectrum of activity against class A, class C, and some class D β-lactamases. Unique among previously available β-lactamase inhibitors (clavulanic acid, tazobactam, and sulbactam), the DBOs do not contain a β-lactam group. Inhibition is accomplished by the formation of a covalent acyl-enzyme complex between the active-site serine of the β-lactamase and the 8-membered cyclooctane ring of the DBO. Interestingly, deacylation typically occurs through a reversible mechanism that regenerates an intact molecule of avibactam, allowing for inhibition of further enzymes. ^22,97,98 KPC-2 and metallo-β-lactamases possess the ability to hydrolyze avibactam; however, the rate of hydrolysis is very slow.^85,99

REPORTS AND MECHANISMS OF RESISTANCE: CEFTAZIDIME-AVIBACTAM

Table 4 lists the existing reports of resistance to ceftazidime-avibactam described to date. Phenotypic resistance to ceftazidime-avibactam appears to be driven largely by amino acid substitutions and deletions to the KPC carbapenemase found in Enterobacterales. These changes were reported mostly in case studies and laboratory selection experiments. Alarmingly, the first case report of ceftazidime-avibactam resistance in a K pneumoniae strain with bla_KPC-3 was in the same year that ceftazidime-avibactam was released.^100,101

Ceftazidime-Avibactam–Resistant KPC Variants

Substitutions in KPC that lead to ceftazidime-avibactam resistance tend to cluster into 1 of 2 regions of the enzyme: substitutions, insertions, and deletions in the Ω-loop, residues 164 to 179 in class A β-lactamases, or insertions in the B3–4 β-strands and adjacent helices (Fig. 7, see Table 4). The importance of the Ω-loop of KPC in ceftazidime-avibactam resistance was revealed first in the summer of 2015 shortly after the release of the combination.¹⁰² By exploiting the knowledge that evolution of the Ω-loop in β-lactamases is the Achilles heel for ceftazidime’s antimicrobial activity, ^16–19 several Ω-loop variants (R164A, R164P, D179A, D179Q, and D179N) of KPC-2 were tested and found resistant to ceftazidime-avibactam (MIC range: 16–64 μg/mL). Additionally, in vitro selection experiments conducted using KPC-3-producing Enterobacterales resulted in the selection of the D179Y variant of KPC-3 among other alterations (see Table 4) that led to ceftazidime-avibactam resistance, further exposing the Ω-loop as a weakness for this combination.¹⁰³ Subsequently, reports of ceftazidime-avibactam resistance began to emerge during treatment of patients with infections caused by carbapenem-resistant Enterobacterales carrying the D179Y variant of KPC-3, which elevated the ceftazidime-avibactam MICs from 2 μg/mL to 4 μg/mL prior to the start of treatment up to 64 μg/mL to greater than 256 μg/mL after treatment^104,105 (see Table 4). Concomitant with the development of ceftazidime-avibactam resistance, bacteria producing the D179Y variant lose carbapenem resistance. Importantly, the tyrosine substitution at 179 was shown to revert back to aspartic acid when grown in the presence of a carbapenem; thus, KPC regains its ability to hydrolyze carbapenems when exposed to a carbapenem.^106–109

Fig. 7.

KPC-2 crystal structure highlighting the major active site motifs (blue: Ω-loop, R₁₆₄-D₁₇₉; magenta: S₇₀X₇₁X₇₂K₇₃ motif; dark green: S₁₃₀D₁₃₁N₁₃₂ loop; and bright green: K₂₃₄T₂₃₅G₂₃₆ motif) (left) and the location of the amino acid substitutions, insertions, and deletions (cyan: insertion after residue; yellow: substitution; orange: deletion or substitution; red: deletion; green: insertion or substitution; and purple deletion or insertion) that confer ceftazidime-avibactam resistance (right).

Another group found this reversion phenotype with other Ω-loop amino acid substitutions (D176Y, P174L, and R164S) in KPC that also caused elevated ceftazidime-avibactam MICs¹¹⁰ In 1 case, the 179 substitution reverted to aspartic acid and ceftazidime-avibactam resistance was maintained (MIC: 12 μg/mL) through amplification of bla_KPC-2 and loss of OmpK35 and OmpK36 when treating the patient with meropenem/polymyxin B.¹⁰⁸ Moreover, the addition of a polymyxin, colistin, to ceftazidime-avibactam does not prevent the emergence of resistance to ceftazidime-avibactam by KPC-producing Enterobacterales.¹¹¹ Another study assessed population diversity and found that wild-type KPC-3 and KPC-3 D179Y coexisted as a mixed population.¹¹² Based on these observations, a case can be made that carbapenem monotherapy should not be considered as a therapeutic regimen against ceftazidime-avibactam–resistant KPC-producing Enterobacterales despite their carbapenem-susceptible phenotype.¹¹³ The clinical risk factors associated with the potential for KPC-producing Enterobacterales to acquire ceftazidime-avibactam resistance during treatment include pneumonia and renal replacement therapy.¹¹⁴ Likely, the concentration of the drug combination at the infection site does not remain above the MIC for a sufficient time and resistant variants are selected; therapeutic drug monitoring and proper dosage selection may be helpful in these cases.^115,116 This leads to speculation that perhaps the dosing of ceftazidime avibactam should be modified in certain cases.

In addition to the D179Y variant of KPC-3, other variants, D179Y T243M, V240G, A177E D179Y, Δ166–167, L7P D179Y T243M, and V240A, also began to emerge in the clinic.^104,117,118 The D179Y variant,^{103,104,106,109,110,117,119–123} however, continues to be the most commonly reported substitution leading to ceftazidime-avibactam resistance and also has emerged in KPC-2.¹⁰⁸ The KPC-2 D179Y variant also was identified in a P aeruginosa strain in Chile, where ceftazidime-avibactam was never used.¹²⁴ Importantly, this study was evaluating a rapid immunochromatographic test for detection of KPC and the variant was not identified by this test or Carba-NP testing.¹²⁴ Also, in the Ω-loop, L169P occurred in KPC-2 during the treatment of a patient for VABP; the cloned KPC-2 L169P variant expressed in E coli DH5α possessed an MIC of 4 μg/mL, while the parent clinical strain’s MIC was 16 μg/mL.¹²⁵ Subsequently, another L169P variant in conjunction with an A172T substitution emerged in KPC-3 during the treatment of an IAI caused by K pneumoniae; in the same study, the patient also was infected with 2 ceftazidime-avibactam–resistant K pneumoniae carrying the KPC-3 D179Y variant and a KPC-3 A172T variant and became colonized by a fourth ceftazidime-avibactam–resistant KPC-3 A172T T243A variant.¹²² The rapidly converting and changing phenotypes of these KPC-3 variants is concerning. Other KPC-2 variants that surfaced during treatment, include 1 that acquired 2 insertions (E and L) between Ω-loop residues 166–167 and S-E-A-V between the C-terminal α-helix residues 278–281 that resulted in a ceftazidime-avibactam MIC of 128 μg/mL.¹¹⁷ A deletion of residues G242-T243 in the B4 β-strand of KPC-2 and KPC-3 (KPC-14 and KPC-28, respectively) resulted in low-level resistance to ceftazidime-avibactam (MICs: 12–24 μg/mL) due to increased catalytic efficiency toward ceftazidime mediated by lower K_m values; inhibition kinetics revealed that avibactam possessed similar IC₅₀ values (range: 107–586 nM) against all variants tested.¹²⁶

Two laboratory studies selected for ceftazidime-avibactam–resistant mutants using various Enterobacterales parent strains producing KPC-3 and found many substitutions that conferred resistance to ceftazidime-avibactam.^103,109 The D179Y, D163G (a location before the Ω loop), and P174L substitutions were identified in both studies; however, only D179Y emerged in the clinic. One study further examined if imipenem susceptibility was affected by the acquisition of ceftazidime-avibactam resistance.¹⁰⁹ The KPC-3 D163G, R164S, N170D, A172S, A172T, A172P, P174L, G175V, Y241N, and T243M variants, along with a KPC-3 ΔV240 variant, and several KPC-3 variants with different insertions in the B5 β-strand and subsequent loop residues (263–278) maintained imipenem resistance (MICs: ≥32 μg/mL), while correspondingly acquiring ceftazidime-avibactam resistance (range: 32 to >256 μg/mL).¹⁰⁹ These data further exemplify the need to screen KPC-producers against carbapenems and ceftazidime-avibactam.

Unfortunately, these gain-of-function observations were not limited to the laboratory. The B5 β-strand seems capable of absorbing large changes leading to increases in ceftazidime-avibactam MICs, with an insertion of NH₂-A-V-Y-T-R-A-P-N-K-D-D-K-H-S-E-CO₂ in the B5 β-strand between residues 261 and 262 of KPC-2 raising MICs from 1 μg/mL to greater than 16 μg/mL; this KPC-2 variant surfaced during treatment of a patient for bacteremia due to K pneumoniae.¹²⁷ In addition, another insertion in the B5 β-strand of PNK between K270 and D271 of KPC-3 in a K pneumoniae strain was obtained from a rectal swab of a patient and resulted in a ceftazidime-avibactam MIC of greater than 128 μg/mL.¹²⁸ This KPC-3 variant was purified for kinetic characterization and the results implicated a lower K_d for the variant with ceftazidime as the primary driver for resistance, as conversely the K_i for avibactam increased 6-fold from KPC-3.¹²⁸

The Mechanism Behind the Resistance: KPC D179N, an Ω-Loop Variant

Among the most-studied β-lactamase variants leading ceftazidime-avibactam resistance is the D179N variant of KPC-2. The D179 residue forms a salt bridge with R164 in wild-type KPC-2 (Fig. 8). When the negatively charged aspartic acid residue at 179 (D) is changed to a polar asparagine (N) and expressed in E coli DH10 B, the ceftazidime-avibactam MIC increases from 1 μg/mL to 16 μg/mL.¹²⁹ Steady-state kinetic characterization of the purified KPC-2 D179N variant revealed that ceftazidime is hydrolyzed at a slower rate with the variant compared with wild-type KPC-2. The apparent K_m value for ceftazidime with the KPC-2 D179N variant, however, was 130 μM compared with 3500 μM with wild-type KPC-2. These kinetic observations suggest that the KPC-2 D179N variant forms more favorable interactions with ceftazidime than wild-type KPC-2 does. The inhibition by avibactam of the KPC-2 D179N variant was not significantly altered compared with wild-type KPC-2 (acylation rates: 38,000 M⁻¹s⁻¹ vs 17,000 M⁻¹s⁻¹, respectively).¹⁰² Electrospray-ionization mass spectrometry used to capture β-lactamase adducts revealed the unique trapping phenotype of the KPC-2 D179N variant. Acyl-enzyme adducts were detected when all β-lactams (eg, ceftazidime, ceftolozane, and imipenem) were incubated with the KPC-2 D179N variant but not with wild-type KPC-2, which presumably hydrolyzed these substrates. Moreover, when the β-lactamases were incubated with equimolar concentrations of β-lactam and avibactam, the KPC-2 D179N variant preferentially bound the β-lactam, whereas KPC-2 favored avibactam. Molecular modeling revealed that the flexibility and mobility of the Ω-loop were increased in the KPC-2 D179N variant due to disruption of the salt bridge with R164; this mobility likely allows ceftazidime to interact more favorably with the active site of the variant (see Fig. 8A, B). In addition, the catalytic residue S70 and the general base E166 were repositioned, thus allowing for a longer-lasting acyl-enzyme complex with the variant and the observed trapping phenotype (see Fig. 8C, D).¹²⁹

Fig. 8.

Molecular modeling and 500-ns molecular dynamic simulation revealed the flexibility and mobility of the Ω-loop was increased in the (B) KPC-2 D179N variant due to disruption of the salt bridge with R164; mobility is RMSD of (A) 2 Å in KPC-2 versus (B) 10 Å for D179N variant. (C) In KPC-2, the R164 residue forms a salt bridge with D179 and hydrogen bonding network with a water molecule (W1). (D) The substitution D179N disrupts the salt bridge, and the nucleophilic S70 and the general base, E166 are repositioned, which results in the repositioning of the catalytic water (W2) and the formation of a longer-lasting acyl-enzyme complex with the variant and ceftazidime.

In addition to the analysis of the KPC-2 D179N variant, the KPC-2 D179Y variant was investigated by another group and they found the variant to have a greater than 43-fold decrease in K_m toward ceftazidime and a greater than 1000-fold decrease in k_cat compared with wild-type KPC-2.¹⁰⁶ These data suggest that the variant can form favorable interactions with ceftazidime more readily than wild-type but is not able to hydrolyze ceftazidime; this is similar to the observation with the KPC-2 D179N variant.¹⁰⁶ Conversely, the acylation rate of avibactam toward the KPC-2 D179Y variant was decreased significantly compared with wild-type (0.4 M⁻¹s⁻¹ vs 29,000 M⁻¹s⁻¹, respectively). The KPC-2 D179Y variant also appears to trap ceftazidime but is not effectively inhibited by avibactam.

Avibactam-Resistant Variants of KPC

Oxapenem, sulfones, and DBO β-lactamase inhibitors follow a similar reaction pathway toward acyl-enzyme formation. Indeed, substitutions (eg, S130G, K234R, and R220M) that have an impact on inhibition by traditional inhibitors, such as clavulanic acid, also effect the ability of avibactam to inhibit β-lactamases.¹³⁰ The S130G substitution in KPC-2 resulted in the inability of avibactam to effectively acylate the enzyme. A subsequent report revealed the importance of the N132 residue in KPC-2 for acylation by avibactam; the N132G mutant was also unable to be acylated by avibactam.¹³¹ Fortunately, these substitutions also reduced KPC-2’s hydrolysis of β-lactams; thus, the contribution toward ceftazidime-avibactam resistance was limited. As KPC enzymes continue to evolve, however, the impact of these inhibitor-resistant substitutions may emerge.

Contributions of Other Class A Enzymes

Amino acid substitution of P167S in the Ω-loop of CTX-M-14 occurring in combination with T264I and OXA-48 resulted in a ceftazidime-avibactam MIC of 32 μg/mL for K pneumoniae.¹³² The role of the Ω-loop in expanding the spectrum of CTX-M-15 enzymes against ceftazidime-avibactam was assessed and the combination of L169Q (Ω-loop) and S130G (SDN loop) in CTX-M-15 resulted in an MIC of 16 μg/mL, when expressed in E coli; the purified CTX-M-15 S130 G L169Q variant hydrolyzed ceftazidime efficiently and was not inhibited by avibactam (IC₅₀ >50 mM).¹³³ K oxytoca with bla_TEM-1 and bla_SHV-12 tested nonsusceptible to ceftazidime-avibactam in a large surveillance study.¹³⁴ In other large surveillance studies, P aeruginosa isolates producing class A PER, GES, or VEB β-lactamases demonstrated reduced susceptibility to ceftazidime-avibactam^76,135–137; however, some of these isolates were not evaluated for other potentially contributing mechanisms.¹³⁵ Another study found that the production of GES-5 and PER-1 in P aeruginosa did result in ceftazidime-avibactam resistance; however, when these bla genes were cloned and expressed in E coli TOP10, the MICs were lowered to 0.5 μg/mL and 16 μg/mL, respectively.³¹

Similarly, the P aeruginosa background causes elevated MICs against these agents, as was seen with ceftolozane-tazobactam. Low-level resistance in E coli is amplified when both GES-19 and GES-26 are introduced in E coli TG1; the ceftazidime-avibactam MIC increased from 0.5 μg/mL to 256 μg/mL, which was comparable to the parent P aeruginosa MICs of 128 to greater than 256 μg/mL.⁷⁷ Recently, 2 different patients in Greece acquired K pneumoniae producing a VEB-1 K234R variant that demonstrated resistance to ceftazidime-avibactam (MICs: 32–128 μg/mL).¹³⁸

Resistance in Enterobacterales AmpCs

Enterobacterales AmpCs can acquire resistance to ceftazidime-avibactam. Single amino acid substitutions were selected for in Enterobacter cloacae AmpC (G156R and G156D) and C freundii AmpC (R148H, R148P, and N346Y) that raised MICs to ceftazidime-avibactam from 0.5 μg/mL for the parent strains to 16 μg/mL to 32 μg/mL for the selected isolates.¹³⁹ Moreover, a deletion of 289–294 in the Enterobacter cloacae AmpC resulted in a ceftazidime-avibactam MIC of 64 μg/mL.¹⁴⁰ Evidence suggests that deletions in the vicinity of residue 290 are the result of enlargement in the R2 binding pocket allowing for β-lactams with larger R2 groups, such as ceftazidime, to be better accommodated.¹⁴⁰

Ceftazidime-Avibactam Resistance of Pseudomonas-Derived Cephalosporinase Variants

Laboratory selection experiments in P aeruginosa identified several changes in PDC (ΔR210-E219, ΔK204a-G222, and ΔD217-Y221) that resulted in ceftazidime-avibactam resistance.¹⁴¹ The ΔD217-Y221 in PDC increased the baseline MIC from 8 μg/mL to 256 μg/mL for ceftazidime-avibactam when expressed in P aeruginosa.¹⁴¹ Purification of the wild-type and variant PDCs revealed that the ΔD217-Y221 variant’s k_cat for ceftazidime increased by 650-fold and IC₅₀ value for avibactam increased by 25-fold. Thus, resistance in this variant was due to increased turnover of ceftazidime as well as reduced inhibition by avibactam. Subsequently, during the selection of ceftolozane-tazobactam–resistant variants, cross-resistance to ceftazidime-avibactam was revealed. Several of the same substitutions in PDC T96I,⁵² G156D,^{57,60,141,142} including Ω-loop substitutions V211A^50,54 and E219K,⁵² combinations Q128R V211T S279T, and Q128R V211A G220S,⁵⁴ and ΔP208-G214⁵⁰ and ΔG202-E219,⁵² that result in resistance to ceftolozane-tazobactam have been reported to also lead to ceftazidime-avibactam resistance in P aeruginosa. Cross-resistance in P aeruginosa to ceftazidime-avibactam and ceftolozane-tazobactam is highly alarming. A large surveillance study also revealed many PDC variants present in P aeruginosa led to resistance to ceftazidime-avibactam; however, the contribution of these variants toward this resistance was not validated.¹³⁶ Acquisition of novel non-PDC AmpCs in P aeruginosa, PAC-1, or FOX-4 also resulted in resistance to ceftazidime-avibactam.^34,74 Moreover, the ability of P aeruginosa to become ceftazidime-avibactam–resistant was found more pronounced in a hypermutator background.¹⁴²

Other Considerations

In Enterobacterales, loss of outer membrane proteins (porins) did not result in resistance to ceftazidime-avibactam even in KPC-producing, AmpC-producing, and/or ESBL-producing strains.^143,144 Strains carrying KPC-2, ESBLs (ie, TEM, SHV, or CTX-M), and ompK36 porin mutations, however, demonstrated statistically significant higher MICs toward ceftazidime-avibactam.¹⁴³ Moreover, overexpression of bla_KPC in conjunction with either loss of OmpK35 and OmpK36 and/or production of ESBLs has been reported to contribute to ceftazidime-avibactam resistance^{119,123,145–148}; in a patient who failed on ceftazidime-avibactam treatment, the ceftazidime-avibactam MICs increased from 4 μg/mL to 32 μg/mL after therapy. Loss of OmpK35 and OmpK36 in concurrence with the production of the DHA-1 class C β-lactamase in K pneumoniae also elevated ceftazidime-avibactam MICs to 16 μg/mL,⁴¹ as did loss of porins and production of CTX-M-15 and OXA-1 in K pneumoniae.¹⁴⁹

In 3 large surveillance studies of 10,998 Klebsiella species, 6209 Enterobacterales, and 36,380 Enterobacterales, small subsets of isolates (n = 16, n = 5, and n = 14, respectively) were resistant to ceftazidime-avibactam and the mechanisms for most of these resistant strains could not be determined.^150–152 The investigators of 1 study proposed that potentially these isolates may have novel modifications of β-lactamases or PBP sequences or changes in drug efflux levels.¹⁵⁰ Indeed, 2 different 4–amino acid insertions in PBP3 found in 3 different E coli isolates carrying various bla genes possessed elevated ceftazidime-avibactam MICs of 8 μg/mL.^153,154 Contrary to Enterobacterales, out of 7062 P aeruginosa tested in a surveillance study, 272 isolates were resistant to ceftazidime-avibactam also with undefined resistance mechanisms; thus, a higher proportion of P aeruginosa are resistant to ceftazidime-avibactam compared with Enterobacterales.¹⁵⁵ In P aeruginosa, overexpression of bla_PDC as well as efflux and permeability of ceftazidime-avibactam appear to play a role in resistance to the combination.^{63,136,156–159} In a P aeruginosa PAO1 background, however, controlled (eg, ΔampD and ΔdacB) de-repression of wildtype bla_PDC-1, loss of oprD, and/or hyperexpression of efflux pumps (eg, ΔmexR and ΔmexZ) did not have a a significant impact on ceftazidime-avibactam MICs (range: 1–4 μg/mL).¹⁵⁶ In vitro selection experiments using P aeruginosa strain PA14 revealed the mutations in dnaJ, pepA, ctpA, glnD, flgF, pcm, spoT, and genes encoding an unidentified 2-component system and efflux pump component also effected resistance to ceftazidime-avibactam (MICs: ≥256 μg/mL); the exact mechanisms are not well understood.¹⁶⁰

The Impact of Class D Oxacillinases

Except for OXA-48, most class D β-lactamases are not inhibited well by avibactam; however, as long as these enzymes are poor ceftazidimases, ceftazidime-avibactam will restore susceptibility, with ceftazidime doing the heavy lifting. Mutations in genes encoding OXA enzymes that extend their profile to ceftazidime have been reported to cause ceftazidime-avibactam resistance.^52,78,79 When the D149 residue in OXA-2 was duplicated, the ceftazidime-avibactam MIC for P aeruginosa PAO1 expressing OXA-2 versus the variant enzyme increased from 1 μg/mL to 32 μg/mL.⁷⁸ The expression of other bla genes (eg, bla_SHV and bla_CTX-M) also has been shown to result in elevated ceftazidime-avibactam MICs (16–64 μg/mL) in Enterobacterales producing OXA-48.¹⁶¹ In vitro laboratory selection on ceftazidime-avibactam using E coli MG1655 expressing bla_OXA-48 revealed that substitutions of P68A and Y211S in OXA-48 elevated ceftazidime-MICs and the OXA-48 P68A Y211S variant possessed an approximately 6-fold increase in K_i for avibactam.¹⁶²

Overcoming the Limitation Incurred from Class B Metallo-β-Lactamases

Not surprising, given that they fall outside the target activity of avibactam, the class B metallo-β-lactamases commonly are associated with high-level resistance to ceftazidime-avibactam. Notably, clinical case reports⁷⁷ and laboratory testing^163–165 suggest that the addition of aztreonamto ceftazidime-avibactammay be able to overcome resistance caused by the coproduction of metallo-β-lactamases and serine β-lactamases.

NEWER β-LACTAM AND β-LACTAMASE INHIBITOR COMBINATIONS: MEROPENEM-VABORBACTAM

In 2017, the FDA approved meropenem-vaborbactam (vaborbactam previously was known as RPX-7009) for the treatment of adult patients with cUTI, including pyelonephritis. ¹⁶⁷ The combination also is undergoing clinical testing for use in pediatric patients with severe infections (NCT02687906) as well as in patients with HABP/VABP (NCT03006679).

Meropenem-vaborbactam demonstrates antimicrobial activity against E coli, K pneumoniae, a n d Enterobacter cloacae complex (Table 5) (Meropenem-Vaborbactam PI). Limitations for this combination against indicated organisms include those that carry class B metallo-carbapenemases (eg, VIM, NDM, and IMP) and/or class D OXA β-lactamases that are not susceptible to inhibition by meropenem or vaborbactam.

Table 5.

Clinical indications for the use of meropenem-vaborbactam

Indication	Indicated Bacteria	Patient Population	Clinical Trials
cUTI, including pyelonephritis	E coli, K pneumoniae, and Enterobacter cloacae species complex	Adult	TANGO I168 NCT02166476

Data from Melinta Therapeutics, Inc. VABOMERE (Meropenemand and Vaborbactam) for Injection, for Intravenous Use. Lincolnshire, IL 60069 USA; 2019.

Meropenem has been in use in the United States since 1996 and is a carbapenem β-lactam antibiotic noted for its stability in the presence of human dehydropeptidase I (an enzyme which quickly metabolizes imipenem).¹⁶⁹ Unfortunately, meropenem is susceptible to hydrolysis by class A carbapenemases, such as KPC; class B metallo-β-lactamases, such as NDM, VIM, and IMP; and class D OXA carbapenemases (eg, OXA-48). In addition, loss of outer membrane proteins (eg, OmpK35 and OmpK36 in K pneumoniae) and increases in efflux pump production (eg, AcrAB-TolC) effect its penetration.¹⁷⁰

Vaborbactam is a cyclic boronate-based β-lactamase inhibitor designed to be selective for β-lactamases over other serine hydrolase enzymes. Intended to have a carbapenem partner from early development, the focus was placed on inhibiting the class A carbapenemase, KPC.¹⁷¹ Vaborbactam is a potent inhibitor of KPC (K_{i app} = 69 nM) and other class A β-lactamases (CTX-M, SHV, and TEM) as well as class C β-lactamases (eg, CMY-2, P99) but not class D serine or class B metallo-β-lactamases.¹⁷²

REPORTS AND MECHANISMS OF RESISTANCE: MEROPENEM-VABORBACTAM

In Table 6, the reports of resistance to meropenem-vaborbactam available to date are presented. Despite 2 years on the market, clinical case reports of meropenem-vaborbactam resistance remain elusive in the literature. Whether this is indicative of the properties of the combination itself or the result of limited use and careful screening for susceptibility on the part of clinicians remains to be seen.

Table 6.

Mechanisms of resistance to meropenem-vaborbactam

Type	Organism	Mechanism(s)	Minimum Inhibitory Concentraiton of Isolatea	Reference
Survey	K pneumoniae	KPC, loss of ompK37 and up-regulation of AcrAB-TolC	16	177
Survey	K pneumoniae	KPC-2, TEM-181, SHV-11, loss of ompK35 and ompK36	16	173
Survey	K pneumoniae	KPC-2, TEM-1, SHV-11, SHV-12, loss of ompK35 and ompK36	32	173
Survey	K pneumoniae	KPC, loss of ompK35 and ompK36	32	174
Survey	K pneumoniae	KPC-2, SHV, TEM, OXA-10, loss of ompK35 and ompK36	64	178
Survey	K pneumoniae	KPC-3, SHV-11, SHV-12, loss of ompK35 and ompK36	16	178
Survey	K pneumoniae	KPC-3, SHV-11, loss of ompK35, ompK36, and ompK37	256	123
Survey	K pneumoniae	KPC-3, SHV-11, TEM1a, OXA-9, loss of ompK35, ompK36, and ompK37	256	123

Vaborbactam is maintained at 8 μg/mL when in combination with meropenem

E coli, K pneumoniae, and Enterobacter cloacae complex breakpoint: resistant ≥16 μg/mL.⁸¹

^aThe MIC values for the isolate represent the MIC values obtained for either a clinical isolate obtained from a patient in a case study or part of a surveillance study or a laboratory-selected strain.

Data from Clinical and Laboratory Standards Institute (CLSI). M100: Performance Standards for Antimicrobial Susceptibility Testing. 30th ed.; 2020.

Enterobacterales Resistant to Meropenem-Vaborbactam

Unlike ceftolozane-tazobactam and ceftazidime-avibactam, reports of strains producing β-lactamase variants resistant to meropenem-vaborbactam are only now beginning to be reported. Moreover, meropenem-vaborbactam was shown to be effective against a case of bacteremia caused by a ceftazidime-avibactam–resistant K pneumoniae producing a KPC-2 D179Y variant.¹²⁰ Likely, the change from a cephalosporin β-lactam partner to a carbapenem β-lactam partner is a significant factor for the differences observed in the resistance patterns. The use of a carbapenem partner in a β-lactam–β-lactamase inhibitor combination, however, also presents its own challenges as resistance due to increased efflux and decreased permeability of carbapenems is more problematic than with cephalosporins in gram negatives.¹⁷⁰ In addition to carbapenems using porins for bacterial cell entry, vaborbactam also was found to traverse OmpK35 and OmpK36 in K pneumoniae.¹⁷² Thus, permeability is likely the largest hurdle for meropenem-vaborbactam efficacy. Resistance to meropenem-vaborbactam largely was reported in strains of K pneumoniae producing KPC with loss of expression of OmpK35, OmpK36, and/or OmpK37—mutations in porin genes that result in the production of partially functioning porins (eg, duplication of GD at positions 134 and 135 in OmpK36) also elevated meropenem-vaborbactam MICs.^{123,173–178} Increased expression of acrAB and/or bla_KPC additionally was reported. ^123,176,177 In vitro selection of K pneumoniae producing KPC-2 on meropenem-vaborbactam revealed that the primary resistance mechanisms toward meropenem-vaborbactam were loss of OmpK36 as well as increased copy number of bla_KPC.¹⁷³ One report revealed that the emergence of meropenem-vaborbactam nonsusceptibility (MIC: 8 μg/mL) due to loss of ompK36 during treatment of a patient for bacteremia due to K pneumoniae producing KPC-3.¹⁷⁹ On a promising note, at least 1 study demonstrated synergy between meropenem-vaborbactam and aztreonam in the treatment of Enterobacterales carrying a metallo-β-lactamases.¹⁶⁵

NEWER β-LACTAM AND β-LACTAMASE INHIBITOR COMBINATIONS: IMIPENEM-CILASTATIN-RELEBACTAM

Approved by the FDA in 2019, imipenem-cilastatin-relebactam is indicated for adult use in treating cIAIs and cUTIs, including pyelonephritis. The combination also is being evaluated for use in severe gram-negative infections in pediatric patients (NCT03969901 and NCT03230916) and for HABP/VABP in adults (NCT03583333) and was studied for bacterial pneumonia more broadly (NCT02493764). The combination demonstrates antimicrobial activity against a variety of gram-negative pathogens, including the anaerobes Bacteroides spp (Table 7).¹⁸⁰ Limitations of imipenem-cilastatin-relebactam against organisms for which it is approved to treat include Enterobacterales or P aeruginosa that carry class B metallo-carbapenemases (eg, VIM, NDM, and IMP) or class D OXA β-lactamases that are not susceptible to inhibition by imipenem or relebactam.¹⁸¹

Table 7.

Clinical indications for the use of imipenem-cilastatin-relebactam

Indication	Indicated Bacteria	Patient Population	Clinical Trials
cIAI	Enterobacter cloacae, E coli, K aerogenes, K pneumoniae, and P aeruginosa	Adult	RESTORE- IMI 1182 NCT02452047
cUTI, including pyelonephritis	Bacteroides caccae, Bacteroides fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, C freundii, Enterobacter cloacae, E coli, Fusobacterium nucleatum, K aerogenes, K oxytoca, K pneumoniae, Parabacteroides distasonis, and P aeruginosa	Adult	RESTORE-IMI 1182 NCT02452047

Data from Merck & Co., Inc. RECARBRIO (Imipenem, Cilastatin, and Relebactam) for Injection, for Intravenous Use. Whitehouse Station, NJ 08889 USA.; 2019.

Imipenem-cilastatin originally approved by the FDA in November 1985 and was the first carbapenem in clinical use. The combination brings together a potent carbapenem antibiotic with an inhibitor of human renal dehydropeptidase (cilastatin), reducing renal metabolism of imipenem.¹⁸⁰ Unfortunately, imipenem is susceptible to hydrolysis by class A carbapenemases (KPC), class B metallo-β-lactamases (eg, VIM, NDM, and IMP), and class D carbapenemases (OXA-48). Moreover, decreased permeability due to loss of outer membrane porins (OmpK35, OmpK36, and OprD) and/or increases in efflux pump production (AcrAB-TolC and MexAB-OprM) effect its activity.¹⁷⁰

Relebactam is a DBO β-lactamase inhibitor that was chosen from among many similar candidate compounds in a search for inhibitors to potentiate imipenem activity. It was selected for having particularly strong inhibitory activity against both class A and class C β-lactamases, demonstrating highly compatible pharmacokinetics with imipenem, effectiveness in mouse models of imipenem-resistant P aeruginosa and K pneumoniae strains, and favorable results in safety testing.¹⁸³

REPORTS AND MECHANISMS OF RESISTANCE: IMIPENEM-RELEBACTAM

Table 8 lists all the reports of resistance to imipenem-relebactam described to date. On the market in the United States for less than 6 months at the time of this writing, clinical case reports of resistance to imipenem-cilastatin-relebactam are not present in the literature. As with meropenem-vaborbactam, reports of strains producing β-lactamase variants resistant to imipenem-relebactam have not been described. Moreover, the imipenem, carbapenem partner, is susceptible to the same mechanisms of resistance as meropenem; increased efflux and decreased permeability are major barriers for carbapenem activity.¹⁷⁰ Being new to the market, a reasonable assumption is that further studies of resistance mechanisms and clinical case reports of emerging resistance and treatment failure of imipenem-relebactam will begin to emerge in the coming year, but until that time, data remains sparse and care should be taken to not draw any speculative conclusions.

Table 8.

Mechanisms of resistance to imipenem-relebactam

Type	Organism	Mechanism(s)	Minimum Inhibitory Concentration of Isolatea	Reference
Survey	K pneumoniae	KPC, loss of ompK36	8	184
Survey	K pneumoniae	Overexpression of KPC-2, TEM-1, SHV-12, loss of ompK35 and ompK36	512	186
Survey	K pneumoniae	KPC-3 TEM-1/SHV-11, loss of ompK35 and ompK36	8	186
Survey	S marcescens	SME-1	4	190
Survey	P aeruginosa	PDC, loss of oprD	8	184
Survey	P aeruginosa	Hyperexpression of PDC, loss of oprD	8	184
Survey	P aeruginosa	GES-5	>8	53

Relebactam is maintained at 4 μg/mL when in combination with imipenem.⁸¹

P aeruginosa breakpoint: resistant ≥8 μg/mL.

Enterobacterales breakpoint: resistant ≥4 μg/mL.

^aThe MIC values for the isolate represent the MIC values obtained for either a clinical isolate obtained from a patient in a case study or part of a surveillance study or a laboratory-selected strain.

Data from Clinical and Laboratory Standards Institute (CLSI). M100: Performance Standards for Antimicrobial Susceptibility Testing. 30th ed.; 2020.

Enterobacterales Resistant to Imipenem-Relebactam

Resistance to imipenem-relebactam was reported mostly in Enterobacterales due to loss of OmpK35/OmpF and OmpK36/OmpC as well as hyperexpression of bla_KPC.^184–188 For β-lactamase–mediated resistance, to date, 1 isolate of K pneumoniae was resistant to imipenem-relebactam and produced a GES-20,¹⁸⁹ whereas 2 isolates of S marcescens with SME also were reported as resistant.^32,190 The contribution (eg, lack of inhibition by relebactam vs enhance hydrolysis of imipenem) of these β-lactamases toward imipenem-relebactam resistance remains to be established.

Pseudomonas aeruginosa Resistant to Imipenem-Relebactam

Contrary to Enterobacterales, most oprD mutants in P aeruginosa were more susceptible to imipenem-relebactam, despite imipenem using OprD for entry into P aeruginosa. ^181,191,192 A previous study revealed that when oprD is not expressed, bla_PDC must be expressed,¹⁹³ and because bla_PDC is inhibited by relebactam the imipenem-relebactam combination is effective against many oprD mutants even when bla_PDC is overexpressed.^53,183,194 Some P aeruginosa strains with decreased expression of oprD and either wild-type or overexpressed levels of PDC, however, were resistant to imipenem-relebactam (MIC: 8 μg/mL).¹⁸⁴ These somewhat contradictory data may be due to the fact that the baseline MICs of oprD mutants toward imipenem and imipenem-relebactam were higher compared with wild-type P aeruginosa strains^181,184; thus, a fine line between susceptibility and resistance exists in these oprD mutants. Efflux was found to not have an impact on the activity of imipenem-relebactam; an oprD mutant strain of P aeruginosa overexpressing MexAB, MexCD, MexXY, and MexJK possessed imipenem-relebactam MICs between 0.125 μg/mL and 1 μg/mL.^53,195 In 2 surveillance studies, of 17 of 589 and 5 of 42, P aeruginosa were found resistant to imipenem-relebactam (MIC range: 8–32 μg/mL) and the mechanism was not defined.^194,196 As with Enterobacterales, the presence of GES carbapenemases (eg, GES-5 and GES-6) in P aeruginosa was found to result in resistance to imipenem-relebactam.^197,198 Indeed, 11% of imipenem-relebactam–resistant P aeruginosa isolates carried GES β-lactamases; imipenem-relebactam MICs ranged from 8 μg/mL to 32 μg/mL for these isolates.¹⁹⁵

SUMMARY

Although the combination therapies covered in this review are highly effective against large collections of clinical isolates, none is perfect, and all have shortcomings. KPC and PDC, the major resistant determinants in Enterobacterales and P aeruginosa, respectively, are evolving at an unprecedented rate. Perhaps the most important takeaway from this review on the development of resistance to some of the most promising advancements in β-lactam antibiotics from the past decade is a reminder: humanity is locked in a constant battle with bacteria that have a huge evolutionary advantage in the fight. Although every new antibiotic or inhibitor that makes it to market provides new tools for physicians to treat otherwise untreatable infections, resistance seemingly remains inevitable. The release of promising new drugs should be heralded, but everyone from doctors and scientists to pharmaceutical companies to policymakers and to the general public needs to realize and remember to not become complacent and that continued research into resistance mechanisms, stewardship and conservation of existing drugs, and development of novel treatments remain essential in the great war between humans and microbe. Judicious use and extensive laboratory testing are needed to prevent further spread especially as new agents enter the armamentarium.

KEY POINTS.

• The novel β-lactam–β-lactamase inhibitor combinations (ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, and imipenem-relebactam) are a significant advance in the therapeutic armamentarium against multidrug-resistant gram-negative pathogens. Unfortunately, resistance to these very powerful agents is emerging rapidly in clinics.

• Resistance to ceftolozane-tazobactam is mediated largely by amino acid substitutions, insertions, and/or deletions in the chromosomal AmpC, Pseudomonas-derived cephalosporinase (PDC), of P aeruginosa.

• Mutations in bla_KPC are the source of most reports of ceftazidime-avibactam resistance in gram negatives.

• A majority of ceftolozane-tazobactam–resistant variants of PDC are cross-resistant to ceftazidime-avibactam.

• The cephalosporin partners in ceftolozane-tazobactam and ceftazidime-avibactam are the major evolutionary drivers toward resistance in these combinations.

• Permeability and efflux are the primary basis for resistance to meropenem-vaborbactam and imipenem-relebactam.