In vitro potency of xeruborbactam in combination with multiple ß-lactam antibiotics in comparison with other ß-lactam/ß-lactamase inhibitor (BLI) combinations against carbapenem-resistant and extended-spectrum ß-lactamase-producing Enterobacterales.

Recently, several ß-lactam (BL)/ß-lactamase inhibitor (BLI) combinations have entered clinical testing or have been marketed for use, but limited direct comparative studies of their in vitro activity exist. Xeruborbactam (XER, also known as QPX7728), which is undergoing clinical development, is a cyclic boronate BLI with potent inhibitory activity against serine (serine ß-lactamase) and metallo-ß-lactamases (MBLs). The objectives of this study were (i) to compare the potency and spectrum of ß-lactamase inhibition by various BLIs in biochemical assays using purified ß-lactamases and in microbiological assays using the panel of laboratory strains expressing diverse serine and metallo-ß-lactamases and (ii) to compare the in vitro potency of XER in combination with multiple ß-lactam antibiotics to that of other BL/BLI combinations in head-to-head testing against recent isolates of carbapenem-resistant Enterobacterales (CRE). Minimal inhibitory concentrations (MICs) of XER combinations were tested with XER at fixed 4 or 8 µg/mL, and MIC testing was conducted in a blinded fashion using Clinical and Laboratory Standards Institute reference methods. Xeruborbactam and taniborbactam (TAN) were the only BLIs that inhibited clinically important MBLs. The spectrum of activity of xeruborbactam included several MBLs identified in Enterobacterales, e.g., and various IMP enzymes and NDM-9 that were not inhibited by taniborbactam. Xeruborbactam potency against the majority of purified ß-lactamases was the highest in comparison with other BLIs. Meropenem-xeruborbactam (MEM-XER, fixed 8 µg/mL) was the most potent combination against MBL-negative CRE with MIC90 values of 0.125 µg/mL. MEM-XER and cefepime-taniborbactam (FEP-TAN) were the only BL/BLIs with activity against MBL-producing CREs; with MEM-XER (MIC90 of 1 µg/mL) being at least 16-fold more potent than FEP-TAN (MIC90 of 16 µg/mL). MEM-XER MIC values were ≤8 µg/mL for >90% of CRE, including both MBL-negative and MBL-positive isolates, with FEP-TAN MIC of >8 µg/mL. Xeruborbactam also significantly enhanced potency of other ß-lactam antibiotics, including cefepime, ceftolozane, ceftriaxone, aztreonam, piperacillin, and ertapenem, against clinical isolates of Enterobacterales that carried various class A, class C, and class D extended-spectrum ß-lactamases and carbapenem-resistant Enterobacterales, including metallo-ß-lactamase-producing isolates. These results strongly support further clinical development of xeruborbactam combinations.

Intrinsic Antibacterial Activity of Xeruborbactam In Vitro: Assessing Spectrum and Mode of Action.

Xeruborbactam (formerly QPX7728) is a cyclic boronate inhibitor of numerous serine and metallo-beta-lactamases. At concentrations generally higher than those required for beta-lactamase inhibition, xeruborbactam has direct antibacterial activity against some Gram-negative bacteria, with MIC50/MIC90 values of 16/32 µg/mL and 16/64 µg/mL against carbapenem-resistant Enterobacterales and carbapenem-resistant Acinetobacter baumannii, respectively (the MIC50/MIC90 values against Pseudomonas aeruginosa are >64 µg/mL). In Klebsiella pneumoniae, inactivation of OmpK36 alone or in combination with OmpK35 resulted in 2- to 4-fold increases in the xeruborbactam MIC. In A. baumannii and P. aeruginosa, AdeIJK and MexAB-OprM, respectively, affected xeruborbactam's antibacterial potency (the MICs were 4- to 16-fold higher in efflux-proficient strains). In Escherichia coli and K. pneumoniae, the 50% inhibitory concentrations (IC50s) of xeruborbactam's binding to penicillin-binding proteins (PBPs) PBP1a/PBP1b, PBP2, and PBP3 were in the 40 to 70 µM range; in A. baumannii, xeruborbactam bound to PBP1a, PBP2, and PBP3 with IC50s of 1.4 µM, 23 µM, and 140 µM, respectively. Treating K. pneumoniae and P. aeruginosa with xeruborbactam at 1× and 2× MIC resulted in changes of cellular morphology similar to those observed with meropenem; the morphological changes observed after treatment of A. baumannii were consistent with inhibition of multiple PBPs but were unique to xeruborbactam compared to the results for control beta-lactams. No single-step xeruborbactam resistance mutants were obtained after selection at 4× MIC of xeruborbactam using wild-type strains of E. coli, K. pneumoniae, and A. baumannii; mutations selected at 2× MIC in K. pneumoniae did not affect antibiotic potentiation by xeruborbactam through beta-lactamase inhibition. Consistent with inhibition of PBPs, xeruborbactam enhanced the potencies of beta-lactam antibiotics even against strains that lacked beta-lactamase. In a large panel of KPC-producing clinical isolates, the MIC90 values of meropenem tested with xeruborbactam (8 µg/mL) were at least 4-fold lower than those in combination with vaborbactam at 64 µg/mL, the concentration of vaborbactam that is associated with complete inhibition of KPC. The additional enhancement of the potency of beta-lactam antibiotics beyond beta-lactamase inhibition may contribute to the potentiation of beta-lactam antibiotics by xeruborbactam.

The Ultrabroad-Spectrum Beta-Lactamase Inhibitor QPX7728 Restores the Potency of Multiple Oral Beta-Lactam Antibiotics against Beta-Lactamase-Producing Strains of Resistant Enterobacterales.

QPX7728 is a cyclic boronate ultrabroad-spectrum beta-lactamase inhibitor, with potent activity against both serine beta-lactamases and metallo-beta-lactamases. QPX7728 can be delivered systemically by the intravenous (i.v.) or oral route of administration. Oral beta-lactam antibiotics alone or in combination with QPX7728 were evaluated for (i) sensitivity to hydrolysis by various common beta-lactamases and inhibition of hydrolysis by QPX7728, (ii) the impact of non-beta-lactamase-mediated resistance mechanisms on potency of beta-lactams, and (iii) in vitro activity against a panel of clinical strains producing diverse beta-lactamases. The carbapenem tebipenem had stability for many serine beta-lactamases from all molecular classes, followed by the cephalosporin ceftibuten. Addition of QPX7728 to tebipenem, ceftibuten, and amdinocillin completely reversed beta-lactamase-mediated resistance in cloned beta-lactamases from serine enzyme and metalloenzyme classes; the degree of potentiation of other beta-lactams varied according to the beta-lactamase produced. Tebipenem, ceftibuten, and cefixime had the lowest MICs against laboratory strains with various combinations of beta-lactamases and the intrinsic drug resistance mechanisms of porin and efflux mutations. There was a high degree of correlation between potency of various combinations against cloned beta-lactamases and efflux/porin mutants and the activity against clinical isolates, showing the importance of inhibition of beta-lactamase along with minimal impact of general intrinsic resistance mechanisms affecting the beta-lactam. Tebipenem and ceftibuten appeared to be the best beta-lactam antibiotics when combined with QPX7728 for activity against Enterobacterales that produce serine beta-lactamases or metallo-beta-lactamases.

In Vitro Activity of the Ultrabroad-Spectrum Beta-Lactamase Inhibitor QPX7728 in Combination with Multiple Beta-Lactam Antibiotics against Pseudomonas aeruginosa.

QPX7728 is an ultrabroad-spectrum beta-lactamase inhibitor with potent inhibition of key serine and metallo beta-lactamases. QPX7728 enhances the potency of multiple beta-lactams in beta-lactamase-producing Enterobacterales and Acinetobacter spp. In this study, we evaluated the in vitro activity of QPX7728 (QPX; 8 µg/ml) combined with multiple beta-lactams against clinical isolates of Pseudomonas aeruginosa with various beta-lactam resistance mechanisms. Seven hundred ninety clinical isolates were included in this study; 500 isolates, termed a "representative panel," were selected to be representative of the MIC distribution of meropenem (MEM), ceftazidime-avibactam (CAZ-AVI), and ceftolozane-tazobactam (TOL-TAZ) resistance for clinical isolates according to 2017 SENTRY surveillance data. An additional 290 selected isolates ("challenge panel") that were either nonsusceptible to MEM or were resistant to TOL-TAZ or CAZ-AVI were also tested; 61 strains carried metallo-beta-lactamases (MBLs), 211 strains were defective in the carbapenem porin OprD, and 185 strains had the MexAB-OprM efflux pump overproduced based on a phenotypic test. Against the representative panel, susceptibility for all QPX7728/beta-lactam combinations was >90%. For the challenge panel, QPX-ceftolozane (TOL) was the most active combination (78.6% susceptible) followed by equipotent QPX-piperacillin (PIP) and QPX-cefepime (FEP), restoring susceptibility in 70.3% of strains (CLSI breakpoints for the beta-lactam compound alone). For MBL-negative strains, QPX-TOL and QPX-FEP restored the MIC values to susceptibility rates in ∼90% and ∼80% of strains, respectively, versus 68% to 70% for QPX-MEM and QPX-PIP and 63% to 65% for TOL-TAZ and CAZ-AVI, respectively. For MBL-positive strains, QPX-PIP restored the MIC to susceptibility values for ∼70% of strains versus 2% to 40% for other combinations. Increased efflux and impaired OprD had various effect on QPX7728 combination depending on the partner beta-lactam tested. QPX7728 enhanced the potency of multiple beta-lactams against P. aeruginosa, with varied results according to beta-lactamase production and other intrinsic resistance mechanisms.

In Vitro Activity of the Ultra-Broad-Spectrum Beta-Lactamase Inhibitor QPX7728 in Combination with Meropenem against Clinical Isolates of Carbapenem-Resistant Acinetobacter baumannii.

QPX7728 is a recently discovered ultra-broad-spectrum beta-lactamase inhibitor (BLI) with potent inhibition of key serine and metallo-beta-lactamases. QPX7728 enhances the potency of many beta-lactams, including carbapenems, in beta-lactamase-producing Gram-negative bacteria, including Acinetobacter spp. The potency of meropenem alone and in combination with QPX7728 (1 to 16 µg/ml) was tested against 275 clinical isolates of Acinetobacter baumannii (carbapenem-resistant A. baumannii [CRAB]) collected worldwide that were highly resistant to carbapenems (MIC50 and MIC90 for meropenem, 64 and >64 µg/ml). Addition of QPX7728 resulted in a marked concentration-dependent increase in meropenem potency, with the MIC90 of meropenem alone decreasing from >64 µg/ml to 8 and 4 µg/ml when tested with fixed concentrations of QPX7728 at 4 and 8 µg/ml, respectively. In order to identify the mechanisms that modulate the meropenem-QPX7728 MIC, the whole-genome sequences were determined for 135 isolates with a wide distribution of meropenem-QPX7728 MICs. This panel of strains included 116 strains producing OXA carbapenemases (71 OXA-23, 16 OXA-72, 16 OXA-24, 9 OXA-58, and 4 OXA-239), 5 strains producing NDM-1, one KPC-producing strain, and 13 strains that did not carry any known carbapenemases but were resistant to meropenem (MIC ≥ 4 µg/ml). Our analysis indicated that mutated PBP3 (with mutations localized in the vicinity of the substrate/inhibitor binding site) is the main factor that contributes to the reduction of meropenem-QPX7728 potency. Still, >90% of isolates that carried PBP3 mutations remained susceptible to ≤8 µg/ml of meropenem when tested with a fixed 4 to 8 µg/ml of QPX7728. In the absence of PBP3 mutations, the MICs of meropenem tested in combination with 4 to 8 µg/ml of QPX7728 did not exceed 8 µg/ml. In the presence of both PBP3 and efflux mutations, 84.6% of isolates were susceptible to ≤8 µg/ml of meropenem with 4 or 8 µg/ml of QPX7728. The combination of QPX7728 with meropenem against CRAB isolates with multiple resistance mechanisms has an attractive microbiological profile.

In Vitro Activity of the Ultrabroad-Spectrum-Beta-Lactamase Inhibitor QPX7728 against Carbapenem-Resistant Enterobacterales with Varying Intrinsic and Acquired Resistance Mechanisms.

QPX7728 is an investigational ultrabroad-spectrum-beta-lactamase inhibitor (BLI) with potent inhibition of key serine and metallo-beta-lactamases. QPX7728 enhances the potency of many beta-lactams, including carbapenems, in isogenic strains of Gram-negative bacteria producing various beta-lactamases. The potency of meropenem alone and in combination with QPX7728 (tested at fixed concentrations of 1 to 16 µg/ml) was tested against 598 clinical isolates of carbapenem-resistant Enterobacterales (CRE). The panel included 363 strains producing serine carbapenemases, 224 strains producing metallo-beta-lactamases (151 NDM, 53 VIM, and 20 IMP), and 50 strains that did not carry any known carbapenemases but were resistant to meropenem (MIC ≥ 4 µg/ml). The panel was also enriched in strains that had various defects in the major porins OmpK35/OmpF and OmpK36/OmpC. Increasing concentrations of QPX7728 restored the potency of meropenem against CRE, with the meropenem MIC90 decreasing from >64 µg/ml to 0.5 µg/ml for QPX7728 (8 µg/ml). QPX7728 significantly increased the potency of meropenem against CRE with multiple resistance mechanisms; the reduction in the meropenem MIC90 with QPX7728 (8 µg/ml) ranged from 32- to >256-fold. Compared with other beta-lactamase inhibitor combinations, meropenem-vaborbactam, ceftazidime-avibactam, and imipenem-relebactam, meropenem with QPX7728 was the most potent beta-lactam-BLI combination tested against all groups of CRE with multiple resistance mechanisms. Defects in OmpK36 in KPC-producing strains markedly decreased the potency of meropenem with vaborbactam (128-fold increase in the MIC90), whereas only an 8- to 16-fold change was observed with QPX7728 plus meropenem. More than 90% of various CRE subsets (including those with reduced permeability) were susceptible to ≤8 µg/ml of meropenem with QPX7728 at 8 µg/ml or lower. The combination of QPX7728 with meropenem against CRE has an attractive microbiological profile in CRE with multiple resistance mechanisms.

Spectrum of Beta-Lactamase Inhibition by the Cyclic Boronate QPX7728, an Ultrabroad-Spectrum Beta-Lactamase Inhibitor of Serine and Metallo-Beta-Lactamases: Enhancement of Activity of Multiple Antibiotics against Isogenic Strains Expressing Single Beta-Lactamases.

QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor, with potent inhibition of key serine and metallo-beta-lactamases being observed in biochemical assays. Microbiological studies using characterized strains were used to provide a comprehensive characterization of the spectrum of beta-lactamase inhibition by QPX7728. The MICs of multiple antibiotics administered intravenously only (ceftazidime, piperacillin, cefepime, ceftolozane, and meropenem) and orally bioavailable antibiotics (ceftibuten, cefpodoxime, tebipenem) alone and in combination with QPX7728 (4 µg/ml), as well as comparator agents, were determined against panels of laboratory strains of Pseudomonas aeruginosa and Klebsiella pneumoniae expressing over 55 diverse serine and metallo-beta-lactamases. QPX7728 significantly enhanced the potency of antibiotics against strains expressing class A extended-spectrum beta-lactamases (CTX-M, SHV, TEM, VEB, PER) and carbapenemases (KPC, SME, NMC-A, BKC-1), consistent with the beta-lactamase inhibition demonstrated in biochemical assays. It also inhibited both plasmidic (CMY, FOX, MIR, DHA) and chromosomally encoded (P99, PDC, ADC) class C beta-lactamases and class D enzymes, including carbapenemases, such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/72/58). QPX7728 is also a potent inhibitor of many class B metallo-beta-lactamases (NDM, VIM, CcrA, IMP, and GIM but not SPM or L1). Addition of QPX7728 (4 µg/ml) reduced the MICs for a majority of the strains to the level observed for the control with the vector alone, indicative of complete beta-lactamase inhibition. The ultrabroad-spectrum beta-lactamase inhibition profile makes QPX7728 a viable candidate for further development.

Impact of Intrinsic Resistance Mechanisms on Potency of QPX7728, a New Ultrabroad-Spectrum Beta-Lactamase Inhibitor of Serine and Metallo-Beta-Lactamases in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

QPX7728 is an ultrabroad-spectrum boronic acid beta-lactamase inhibitor that demonstrates inhibition of key serine and metallo-beta-lactamases at a nanomolar concentration range in biochemical assays with purified enzymes. The broad-spectrum inhibitory activity of QPX7728 observed in biochemical experiments translates into enhancement of the potency of many beta-lactams against strains of target pathogens producing beta-lactamases. The impacts of bacterial efflux and permeability on inhibitory potency were determined using isogenic panels of KPC-3-producing isogenic strains of Klebsiella pneumoniae and Pseudomonas aeruginosa and OXA-23-producing strains of Acinetobacter baumannii with various combinations of efflux and porin mutations. QPX7728 was minimally affected by multidrug resistance efflux pumps either in Enterobacteriaceae or in nonfermenters, such as P. aeruginosa or A. baumannii Against P. aeruginosa, the potency of QPX7728 was further enhanced when the outer membrane was permeabilized. The potency of QPX7728 against P. aeruginosa was not affected by inactivation of the carbapenem porin OprD. While changes in OmpK36 (but not OmpK35) reduced the potency of QPX7728 (8- to 16-fold), QPX7728 (4 µg/ml) nevertheless completely reversed the KPC-mediated meropenem resistance in strains with porin mutations, consistent with the lesser effect of these mutations on the potency of QPX7728 compared to that of other agents. The ultrabroad-spectrum beta-lactamase inhibition profile, combined with enhancement of the activity of multiple beta-lactam antibiotics with various sensitivities to the intrinsic resistance mechanisms of efflux and permeability, indicates that QPX7728 is a useful inhibitor for use with multiple beta-lactam antibiotics.

Discovery of Cyclic Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of Serine and Metallo-ß-lactamases.

Despite major advances in the ß-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a ß-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.

Absence of TetB identifies minocycline-susceptible isolates of Acinetobacter baumannii.

Minocycline is one of the few options available to treat infections caused by Acinetobacter baumannii. Acquired resistance to minocycline in A. baumannii is associated with presence of the TetB efflux pump. Previous studies suggested that the absence of tetB may predict minocycline minimum inhibitory concentrations (MICs) of ≤4 µg/mL. In this study, a collection of 258 A. baumannii isolates was used to generate MIC frequency distributions for the tetB-positive and -negative sets of isolates. Of the 93 tetB-negative strains, all had minocycline MICs ≤ 4 µg/mL, resulting in a negative predictive value of 100%. Of the 165 tetB-positive strains, 154 had minocycline MICs > 4 µg/mL, resulting in a positive predictive value of 93.3%. In conclusion, this study shows that tetB is highly associated with MICs above the current US Food and Drug Administration (FDA) and Clinical and Laboratory Standards Institute (CLSI) susceptible breakpoint of 4 µg/mL.

Activity of Meropenem-Vaborbactam in Mouse Models of Infection Due to KPC-Producing Carbapenem-Resistant Enterobacteriaceae.

Meropenem-vaborbactam (Vabomere) is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing, carbapenem-resistant Enterobacteriaceae The objective of these studies was to evaluate the efficacy of meropenem alone and in combination with vaborbactam in mouse thigh and lung infection models. Thighs or lungs of neutropenic mice were infected with KPC-producing carbapenem-resistant Enterobacteriaceae, with meropenem MICs ranging from ≤0.06 to 8 mg/liter in the presence of 8 mg/liter vaborbactam. Mice were treated with meropenem alone or meropenem in combination with vaborbactam every 2 h for 24 h to provide exposures comparable to 2-g doses of each component in humans. Meropenem administered in combination with vaborbactam produced bacterial killing in all strains tested, while treatment with meropenem alone either produced less than 0.5 log CFU/tissue of bacterial killing or none at all. In the thigh model, 11 strains were treated with the combination of meropenem plus vaborbactam (300 plus 50 mg/kg of body weight). This combination produced from 0.8 to 2.89 logs of bacterial killing compared to untreated controls at the start of treatment. In the lung infection model, two strains were treated with the same dosage regimen of meropenem and vaborbactam. The combination produced more than 1.83 logs of bacterial killing against both strains tested compared to untreated controls at the start of treatment. Overall, these data suggest that meropenem-vaborbactam may have utility in the treatment of infections due to KPC-producing carbapenem-resistant Enterobacteriaceae.

Potency of Meropenem-Vaborbactam in Lung Surfactant.

This study investigated whether pulmonary surfactant has an effect on the in vitro antibacterial activity of either meropenem alone or meropenem in combination with vaborbactam at a fixed concentration of 8 µg/ml against several Klebsiella pneumoniae carbapenemase (KPC)-producing strains of Gram-negative bacteria. Results showed that the potency of meropenem alone and that of meropenem-vaborbactam were not affected when tested with pulmonary surfactant.

Activity of Simulated Human Dosage Regimens of Meropenem and Vaborbactam against Carbapenem-Resistant Enterobacteriaceae in an In Vitro Hollow-Fiber Model.

The objective of these studies was to evaluate the exposures of meropenem and vaborbactam that would produce antibacterial activity and prevent resistance development in carbapenem-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae strains when tested at an inoculum of 108 CFU/ml. Thirteen K. pneumoniae isolates, three Enterobacter cloacae isolates, and one Escherichia coli isolate were examined in an in vitro hollow-fiber model over 32 h. Simulated dosage regimens of 1 to 2 g of meropenem with 1 to 2 g of vaborbactam, with meropenem administered every 8 h by a 3-h infusion based on phase 1 or phase 3 patient pharmacokinetic data, were studied in the model. A dosage of 2 g of meropenem in combination with 2 g of vaborbactam was bactericidal against K. pneumoniae, E. cloacae, and E. coli strains, with meropenem-vaborbactam MICs of up to 8 mg/liter. When the vaborbactam exposure was adjusted to the levels observed in patients enrolled in phase 3 trials (24-h free AUC, ∼550 mg · h/liter, versus 320 mg · h/liter in the phase 1 studies), 2 g of meropenem with 2 g of vaborbactam was also bactericidal against strains with meropenem-vaborbactam MICs of 16 mg/liter. In addition, this level of vaborbactam also suppressed the development of resistance observed using phase 1 exposures. In this pharmacodynamic model, exposures similar to 2 g of meropenem in combination with 2 g of vaborbactam administered every 8 h by a 3-h infusion in phase 3 trials produced antibacterial activity and suppressed the development of resistance against carbapenem-resistant KPC-producing strains of Enterobacteriaceae.

Meropenem-Vaborbactam Resistance Selection, Resistance Prevention, and Molecular Mechanisms in Mutants of KPC-Producing Klebsiella pneumoniae.

Vaborbactam (formerly RPX7009) is a new ß-lactamase inhibitor based on a cyclic boronic acid pharmacophore with potent inhibitory activity against Klebsiella pneumoniaecarbapenemases (KPC). It has been developed in combination with meropenem. The objective of these studies was to identify the concentrations of both agents associated with the selection or prevention of single-step mutations leading to reduced sensitivity to the combination and to characterize the selected mutations. Eighteen strains of KPC-producing Klebsiella pneumoniae with various degrees of sensitivity to meropenem (MICs, 8 to 512 µg/ml) and meropenem-vaborbactam (MICs, ≤0.06 to 32 µg/ml) and preexisting resistance mechanisms were selected from a worldwide collection of isolates recovered from surveillance studies, emphasizing strains for which MICs were in the upper range of the meropenem-vaborbactam MIC distribution. Meropenem and vaborbactam at 8 µg/ml each suppressed the drug resistance mutation frequency to <1 × 10-8 in 77.8% (14/18) of strains, and all strains were inhibited when the meropenem concentration was increased to 16 µg/ml. Mutants selected at lower drug concentrations showed phenotypes associated with previously described carbapenem resistance mechanisms, including ompK36 inactivation in mutants selected from OmpK36-proficient strains and an increased blaKPC gene copy number in strains with partially functional ompK36 No mutations in the coding region of blaKPC were identified. These data indicate that the selection of mutants with reduced sensitivity to meropenem-vaborbactam from KPC-producing Klebsiella pneumoniae strains is associated with previously described mechanisms involving porin mutations and the increase in the blaKPC gene copy number and not changes in the KPC enzyme and can be prevented by the drug concentrations achieved with optimal dosing of the combination.

Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae.

Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 µg/ml results in meropenem MICs of ≤2 µg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae.

Resistance to Ceftazidime-Avibactam Is Due to Transposition of KPC in a Porin-Deficient Strain of Klebsiella pneumoniae with Increased Efflux Activity.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.