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Wright W. Nichols - One of the best experts on this subject based on the ideXlab platform.
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loss of activity of ceftazidime Avibactam due to mexab oprm efflux and overproduction of ampc cephalosporinase in pseudomonas aeruginosa isolated from patients suffering from cystic fibrosis
International Journal of Antimicrobial Agents, 2018Co-Authors: Hussein Chalhoub, Wright W. Nichols, Yolanda Saenz, Paul M Tulkens, Francoise Van BambekeAbstract:In Pseudomonas aeruginosa (P. aeruginosa) collected from cystic fibrosis (CF) patients, 24% resistance to ceftazidime-Avibactam in isolates negative for carbapenemases and extended-spectrum β-lactamases (ESBLs) has previously been observed. The current study aimed to unravel the underlying mechanism(s). Using the laboratory strain PAO1 and derivatives thereof, with ampC expression induced by a sub-minimum inhibitory concentration (MIC) of imipenem, a higher MIC of ceftazidime-Avibactam was found for those overexpressing MexAB-OprM (quantitative polymerase chain reaction (PCR) of mexA) and, to a lesser extent, MexEF-OprN (PCR of mexE), or without OprD expression (SDS-Page and Coomassie blue staining). This was ascribed to (i) an efflux of Avibactam (efflux mutants) and (ii) a lack of Avibactam penetration (OprD mutants), respectively. We then used 10 CF clinical isolates resistant to ceftazidime (MIC ≥ 128 mg/L) and with (i) variable basal levels of ampC overexpression, (ii) mutations in mexA or mexB inactivating to variable extent the MexAB-OprM transport capacity (assessed by extrusion of N-phenyl-1-naphthylamine [NPN]), and (iii) expression or not of mexE and of OprD porin. The reduction of ceftazidime MIC in the presence of Avibactam was partially lost for isolates with large efflux activity of MexAB-OprM and/or increased ampC expression, but not significantly with mexE expression or lack of OprD (non-parametric and parametric tests). This identified MexAB-OprM as a main Avibactam efflux transporter in P. aeruginosa that, together with ampC overexpression, reduced Avibactam potency. Since about 30% of CF isolates show mutations in MexAB-OprM compromising efflux (Chalhoub, et al. Sci Reports 2017;7:40208), routine susceptibility testing of CF P. aeruginosa with ceftazidime-Avibactam is warranted.
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in vitro susceptibility to ceftazidime Avibactam of carbapenem nonsusceptible enterobacteriaceae isolates collected during the inform global surveillance study 2012 to 2014
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Boudewijn L M De Jonge, James A Karlowsky, Krystyna M Kazmierczak, Daniel F Sahm, Douglas J Biedenbach, Wright W. NicholsAbstract:The activity of ceftazidime-Avibactam was assessed against 961 isolates of meropenem-nonsusceptible Enterobacteriaceae Most meropenem-nonsusceptible metallo-β-lactamase (MBL)-negative isolates (97.7%) were susceptible to ceftazidime-Avibactam. Isolates that carried KPC or OXA-48-like β-lactamases, both alone and in combination with extended-spectrum β-lactamases (ESBLs) and/or AmpC β-lactamases, were 98.7% and 98.5% susceptible to ceftazidime-Avibactam, respectively. Meropenem-nonsusceptible, carbapenemase-negative isolates demonstrated 94.7% susceptibility to ceftazidime-Avibactam. Ceftazidime-Avibactam activity was compromised only in isolates for which carbapenem resistance was mediated through metallo-β-lactamases.
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role of the outer membrane and porins in susceptibility of β lactamase producing enterobacteriaceae to ceftazidime Avibactam
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Jeanmarie Pages, Sabine Peslier, Thomas A Keating, Jeanphilippe Lavigne, Wright W. NicholsAbstract:This study examined the activity of the novel antimicrobial combination ceftazidime-Avibactam against Enterobacteriaceae exhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of Avibactam alone, ceftazidime alone, and ceftazidime-Avibactam against the characterized clinical isolates of Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine β-naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which Avibactam crossed the outer membranes of E. coli and K. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of Avibactam across the outer membrane of E. aerogenes, although other diffusion channels for Avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-Avibactam. Nevertheless, the MICs of ceftazidime-Avibactam (with 4 mg/liter Avibactam) against the ceftazidime-resistant clinical isolates of the three species of Enterobacteriaceae studied were ≤ 8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.
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distinctive binding of Avibactam to penicillin binding proteins of gram negative and gram positive bacteria
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Abdelhamid Asli, Kevin M. Krause, Wright W. Nichols, Eric Brouillette, Francois MalouinAbstract:Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although Avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of Avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, Avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, Avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where Avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, Avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets.
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pharmacodynamics of ceftazidime and Avibactam in neutropenic mice with thigh or lung infection
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Johanna Berkhout, Virna J Schuck, Wright W. Nichols, Maria J Melchers, Seyedmojtaba Seyedmousavi, Claudia M Lagarde, Johan W MoutonAbstract:ABSTRACT Avibactam is a new non-β-lactam β-lactamase inhibitor that shows promising restoration of ceftazidime activity against microorganisms producing Ambler class A extended-spectrum β-lactamases (ESBLs) and carbapenemases such as KPCs, class C β-lactamases (AmpC), and some class D enzymes. To determine optimal dosing combinations of ceftazidime-Avibactam for treating infections with ceftazidime-resistant Pseudomonas aeruginosa, pharmacodynamic responses were explored in murine neutropenic thigh and lung infection models. Exposure-response relationships for ceftazidime monotherapy were determined first. Subsequently, the efficacy of adding Avibactam every 2 h (q2h) or q8h to a fixed q2h dose of ceftazidime was determined in lung infection for two strains. Dosing Avibactam q2h was significantly more efficacious, reducing the Avibactam daily dose for static effect by factors of 2.7 and 10.1, whereas the mean percentage of the dosing interval that free drug concentrations remain above the threshold concentration of 1 mg/liter (% f T>C T 1 mg/liter) yielding bacteriostasis was similar for both regimens, with mean values of 21.6 (q2h) and 18.5 (q8h). Dose fractionation studies of Avibactam in both the thigh and lung models indicated that the effect of Avibactam correlated well with % f T>C T 1 mg/liter. This parameter of Avibactam was further explored for four P. aeruginosa strains in the lung model and six in the thigh model. Parameter estimates of % f T>C T 1 mg/liter for Avibactam ranged from 0 to 21.4% in the lung model and from 14.1 to 62.5% in the thigh model to achieve stasis. In conclusion, addition of Avibactam enhanced the effect of ceftazidime, which was more pronounced at frequent dosing and well related with % f T>C T 1 mg/liter. The thigh model appeared more stringent, with higher values, ranging up to 62.5% f T>C T 1 mg/liter, required for a static effect.
Robert A Bonomo - One of the best experts on this subject based on the ideXlab platform.
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in vitro selection of aztreonam Avibactam resistance in dual carbapenemase producing klebsiella pneumoniae
Journal of Antimicrobial Chemotherapy, 2020Co-Authors: Siqiang Niu, Jie Wei, Chunhong Zou, Kalyan D Chavda, Haifang Zhang, Yiwei Tang, Johann D D Pitout, Robert A Bonomo, Barry N Kreiswirth, Liang ChenAbstract:OBJECTIVES To examine the in vitro selection of aztreonam/Avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. METHODS The MICs of aztreonam were determined with and without Avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. RESULTS We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of Avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/Avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/Avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/Avibactam and aztreonam/Avibactam/ceftazidime. CONCLUSIONS Aztreonam in combination with Avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/Avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.
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ceftazidime Avibactam versus standard of care agents against carbapenem resistant enterobacteriaceae harbouring blakpc in a one compartment pharmacokinetic pharmacodynamic model
Journal of Antimicrobial Chemotherapy, 2018Co-Authors: Katie E Barber, Robert A Bonomo, Jason M Pogue, Henderson D Warnock, Keith S KayeAbstract:Background 'Last-line' antimicrobial usage has promoted the emergence of MDR bacteria. Production of Klebsiella pneumoniae carbapenemases (KPCs) is increasingly common and leads to resistance to most antimicrobials. However, ceftazidime/Avibactam demonstrates activity against KPC-producing strains. Ceftazidime/Avibactam in the empirical setting remains unknown. Methods Strains underwent genetic analysis evaluating blaKPC presence/production and MICs were determined. Four strains were assessed in an in vitro, one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) model for 96 h. The following bolus dosing exposures were tested: 2.5 g of ceftazidime/Avibactam every 8 h, 2 g of meropenem every 8 h, 1.25 mg/kg polymyxin B every 12 h, amikacin 'once-daily dosing' (peak of 70-80 mg/L), tigecycline at 200 mg ×1 dose followed by 100 mg every 12 h, and a drug-free growth control. Results Thirty blaKPC-producing strains were evaluated; 97% of strains were ceftazidime/Avibactam susceptible with MIC50/MIC90 values of 0.38/1.5 mg/L (range 0.032-16 mg/L). Two K. pneumoniae strains, one Klebsiella oxytoca strain and one Citrobacter freundii strain underwent further analysis in PK/PD models. Ceftazidime/Avibactam displayed potent activity with a reduction of 4.23 ± 0.42 cfu/mL from the initial inoculum at 96 h. Against susceptible isolates, amikacin displayed similar activity compared with ceftazidime/Avibactam at 96 h, although this was not demonstrated against all strains. Polymyxin B produced comparable activity to ceftazidime/Avibactam against two strains. Neither meropenem nor tigecycline produced effective killing and were comparable to the drug-free growth control at 96 h. Conclusions blaKPC-producing organisms demonstrated susceptibility to ceftazidime/Avibactam and bactericidal activity was observed in the PK/PD model. Based on these data, ceftazidime/Avibactam is a valuable agent for treating KPC-producing organisms and should be considered for treatment of infections caused by these pathogens.
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in vitro discordance with in vivo activity humanized exposures of ceftazidime Avibactam aztreonam and tigecycline alone and in combination against new delhi metallo β lactamase producing klebsiella pneumoniae in a murine lung infection model
Antimicrobial Agents and Chemotherapy, 2017Co-Authors: Marguerite L Monogue, Robert A Bonomo, Lilian M Abbo, Rossana Rosa, Jose F Camargo, Octavio Martinez, David P NicolauAbstract:The management of infections with New Delhi metallo-beta-lactamase-1 (NDM)-producing bacteria remains clinically challenging given the multidrug resistant (MDR) phenotype associated with these bacteria. Despite resistance in vitro, ceftazidime-Avibactam previously demonstrated in vivo activity against NDM-positive Enterobacteriaceae Herein, we observed in vitro synergy with ceftazidime-Avibactam and aztreonam against an MDR Klebsiella pneumoniae harboring NDM. In vivo, humanized doses of ceftazidime-Avibactam monotherapy resulted in >2 log10 CFU bacterial reduction; therefore, no in vivo synergy was observed.
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ceftazidime Avibactam and ceftolozane tazobactam second generation β lactam β lactamase inhibitor combinations
Clinical Infectious Diseases, 2016Co-Authors: David Van Duin, Robert A BonomoAbstract:Ceftolozane/tazobactam and ceftazidime/Avibactam are 2 novel β-lactam/β-lactamase combination antibiotics. The antimicrobial spectrum of activity of these antibiotics includes multidrug-resistant (MDR) gram-negative bacteria (GNB), including Pseudomonas aeruginosa. Ceftazidime/Avibactam is also active against carbapenem-resistant Enterobacteriaceae that produce Klebsiella pneumoniae carbapenemases. However, Avibactam does not inactivate metallo-β-lactamases such as New Delhi metallo-β-lactamases. Both ceftolozane/tazobactam and ceftazidime/Avibactam are only available as intravenous formulations and are dosed 3 times daily in patients with normal renal function. Clinical trials showed noninferiority to comparators of both agents when used in the treatment of complicated urinary tract infections and complicated intra-abdominal infections (when used with metronidazole). Results from pneumonia studies have not yet been reported. In summary, ceftolozane/tazobactam and ceftazidime/Avibactam are 2 new second-generation cephalosporin/β-lactamase inhibitor combinations. After appropriate trials are conducted, they may prove useful in the treatment of MDR GNB infections. Antimicrobial stewardship will be essential to preserve the activity of these agents.
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activity of ceftazidime Avibactam against isogenic strains of escherichia coli containing kpc and shv β lactamases with single amino acid substitutions in the ω loop
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Marisa L Winkler, Krisztina M Pappwallace, Robert A BonomoAbstract:Objectives: The objective of this study was to explore the activity of ceftazidime and ceftazidime/Avibactam against a collection of isogenic strains ofEscherichiacoli DH10B possessing SHV and KPCb-lactamases containing single amino acid substitutions in the V-loop (residues 164 –179). Methods: Ceftazidime and ceftazidime/Avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 b-lactamase variants that possessed elevated MICs of ceftazidime/Avibactam were selected for further biochemical analyses. Results: Avibactam restored susceptibility to ceftazidime for all V-loop variants of SHV-1 with MICs ,8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/Avibactam .8 mg/L. b-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 b-lactamases indicated that Avibactam effectively inhibited these enzymes. Conclusions: Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in theV-loop were not susceptible to ceftazidime/Avibactam (MICs .8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant b-lactamases that prevent Avibactam from binding to and inhibiting the b-lactamase. As ceftazidime/ Avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important.
Liang Chen - One of the best experts on this subject based on the ideXlab platform.
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in vitro selection of aztreonam Avibactam resistance in dual carbapenemase producing klebsiella pneumoniae
Journal of Antimicrobial Chemotherapy, 2020Co-Authors: Siqiang Niu, Jie Wei, Chunhong Zou, Kalyan D Chavda, Haifang Zhang, Yiwei Tang, Johann D D Pitout, Robert A Bonomo, Barry N Kreiswirth, Liang ChenAbstract:OBJECTIVES To examine the in vitro selection of aztreonam/Avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. METHODS The MICs of aztreonam were determined with and without Avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. RESULTS We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of Avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/Avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/Avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/Avibactam and aztreonam/Avibactam/ceftazidime. CONCLUSIONS Aztreonam in combination with Avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/Avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.
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verification of ceftazidime Avibactam and ceftolozane tazobactam susceptibility testing methods against carbapenem resistant enterobacteriaceae and pseudomonas aeruginosa
Journal of Clinical Microbiology, 2017Co-Authors: Ryan K Shields, Binghua Hao, Ellen G. Press, Barry N Kreiswirth, Liang Chen, Ghady Haidar, Cornelius J Clancy, William A Pasculle, Hong M NguyenAbstract:Ceftazidime-Avibactam and ceftolozane-tazobactam are newly approved agents for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. Resistance to both agents has been described clinically. Susceptibility testing on automated systems is unavailable for either agent. Our objective was to compare the disk diffusion and Etest methods to standard broth microdilution (BMD) methods for testing ceftazidime-Avibactam and ceftolozane-tazobactam against a diverse collection of carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRP) isolates, respectively. Among 74 ceftazidime-Avibactam-susceptible and -resistant CRE isolates, BMD categorical agreement was higher with Etest (96%) than with disk diffusion (72%; P = 0.0003). Twenty-eight percent of ceftazidime-Avibactam-susceptible CRE isolates were classified as resistant by disk diffusion. Results were comparable to those obtained with resistance defined genotypically. Among 72 ceftolozane-tazobactam-susceptible and -resistant CRP isolates, the levels of BMD categorical agreement with disk diffusion and Etest were 94% and 96%, respectively; the only errors identified were minor. Our findings demonstrate that Etest measurements of ceftazidime-Avibactam and ceftolozane-tazobactam susceptibility correlate closely with standard BMD methods, suggesting a useful role clinically. On the other hand, disk diffusion measurements overcalled CRE resistance to ceftazidime-Avibactam. A better understanding of ceftazidime-Avibactam interpretive breakpoints is needed before disk diffusion is used routinely in the clinic. Until clinicians and microbiologists understand Etest and disk diffusion performance at their centers, test results should be interpreted cautiously.
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identifying spectra of activity and therapeutic niches for ceftazidime Avibactam and imipenem relebactam against carbapenem resistant enterobacteriaceae
Antimicrobial Agents and Chemotherapy, 2017Co-Authors: Ghady Haidar, Barry N Kreiswirth, Liang Chen, Cornelius J Clancy, Palash Samanta, Hong M NguyenAbstract:We determined imipenem, imipenem-relebactam, ceftazidime, and ceftazidime-Avibactam MICs against 100 CRE isolates that underwent whole-genome sequencing. Klebsiella pneumoniae carbapenemases (KPCs) were the most common carbapenemases. Forty-six isolates carried extended-spectrum β-lactamases (ESBLs). With the addition of relebactam, imipenem susceptibility increased from 8% to 88%. With the addition of Avibactam, ceftazidime susceptibility increased from 0% to 85%. Neither imipenem-relebactam nor ceftazidime-Avibactam was active against metallo-β-lactamase (MBL) producers. Ceftazidime-Avibactam (but not imipenem-relebactam) was active against OXA-48-like producers, including a strain not harboring any ESBL. Major OmpK36 porin mutations were independently associated with higher imipenem-relebactam MICs (P < 0.0001) and showed a trend toward independent association with higher ceftazidime-Avibactam MICs (P = 0.07). The presence of variant KPC-3 was associated with ceftazidime-Avibactam resistance (P < 0.0001). In conclusion, imipenem-relebactam and ceftazidime-Avibactam had overlapping spectra of activity and niches in which each was superior. Major OmpK36 mutations in KPC-K. pneumoniae may provide a foundation for stepwise emergence of imipenem-relebactam and ceftazidime-Avibactam resistance.
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emergence of ceftazidime Avibactam resistance due to plasmid borne blakpc 3 mutations during treatment of carbapenem resistant klebsiella pneumoniae infections
Antimicrobial Agents and Chemotherapy, 2017Co-Authors: Ryan K Shields, Ellen G. Press, Kalyan D Chavda, Barry N Kreiswirth, Liang Chen, Hong M Nguyen, Shaoji Cheng, Avin C Snyder, Ruchi Pandey, Cornelius J ClancyAbstract:ABSTRACT Ceftazidime-Avibactam is a novel β-lactam/β-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-Avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-Avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-Avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-Avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne bla KPC-3 , which were not present in baseline isolates. bla KPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-Avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and bla KPC cloning into competent Escherichia coli. In rank order, the impact of KPC-3 variants on ceftazidime-Avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ≥4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced ≥4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of bla KPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to bla KPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring bla KPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-Avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.
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evaluation of the in vitro activity of ceftazidime Avibactam and ceftolozane tazobactam against meropenem resistant pseudomonas aeruginosa isolates
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Deanna J Buehrle, Binghua Hao, Ellen G. Press, Ryan K Shields, Brian A Potoski, Barry N Kreiswirth, Liang Chen, Cornelius J Clancy, Ammar Alkrouk, Hong M NguyenAbstract:We compared ceftazidime-Avibactam, ceftolozane-tazobactam, ceftazidime, cefepime, and piperacillin-tazobactam MICs for 38 meropenem-resistant Pseudomonas aeruginosa isolates. No isolates harbored carbapenemases; 74% were oprD mutants. Ceftazidime-Avibactam and ceftolozane-tazobactam were active against 92% of the isolates, including 80% that were resistant to all three β-lactams. Forty-three percent of ceftazidime-Avibactam-susceptible isolates and 6% of ceftolozane-tazobactam-susceptible isolates exhibited MICs at the respective breakpoints. Ceftolozane-tazobactam and ceftazidime-Avibactam are therapeutic options for meropenem-resistant P. aeruginosa infections that should be used judiciously to preserve activity.
Krisztina M Pappwallace - One of the best experts on this subject based on the ideXlab platform.
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switching partners piperacillin Avibactam is a highly potent combination against multidrug resistant burkholderia cepacia complex and burkholderia gladioli cystic fibrosis isolates
Journal of Clinical Microbiology, 2019Co-Authors: Elise T Zeiser, Scott A Becka, Brigid M Wilson, Melissa D Barnes, John J. Lipuma, Krisztina M PappwallaceAbstract:ABSTRACT In persons with cystic fibrosis (CF), airway infection with Burkholderia cepacia complex (Bcc) species or Burkholderia gladioli presents a significant challenge due to inherent resistance to multiple antibiotics. Two chromosomally encoded inducible β-lactamases, a Pen-like class A and AmpC are produced in Bcc and B. gladioli. Previously, ceftazidime-Avibactam demonstrated significant potency against Bcc and B. gladioli isolated from the sputum of individuals with CF; however, 10% of the isolates tested resistant to ceftazidime-Avibactam. Here, we describe an alternative antibiotic combination to overcome ceftazidime-Avibactam resistance. Antimicrobial susceptibility testing was performed on Bcc and B. gladioli clinical and control isolates. Biochemical analysis was conducted on purified PenA1 and AmpC1 β-lactamases from Burkholderia multivorans ATCC 17616. Analytic isoelectric focusing and immunoblotting were conducted on cellular extracts of B. multivorans induced by various β-lactams or β-lactam-β-lactamase inhibitor combinations. Combinations of piperacillin-Avibactam, as well as piperacillin-tazobactam plus ceftazidime-Avibactam (the clinically available counterpart), were tested against a panel of ceftazidime-Avibactam nonsusceptible Bcc and B. gladioli. The piperacillin-Avibactam and piperacillin-tazobactam-ceftazidime-Avibactam combinations restored susceptibility to 99% of the isolates tested. Avibactam is a potent inhibitor of PenA1 (apparent inhibitory constant [Kiapp] = 0.5 μM), while piperacillin was found to inhibit AmpC1 (Kiapp = 2.6 μM). Moreover, piperacillin, tazobactam, ceftazidime, and Avibactam, as well as combinations thereof, did not induce expression of blapenA1 and blaampC1 in the B. multivorans ATCC 17616 background. When ceftazidime-Avibactam is combined with piperacillin-tazobactam, the susceptibility of Bcc and B. gladioli to ceftazidime and piperacillin is restored in vitro. Both the lack of blapenA1 induction and potent inactivation of PenA1 by Avibactam likely provide the major contributions toward susceptibility. With in vivo validation, piperacillin-tazobactam-ceftazidime-Avibactam may represent salvage therapy for individuals with CF and highly drug-resistant Bcc and B. gladioli infections.
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ceftazidime Avibactam in combination with fosfomycin a novel therapeutic strategy against multidrug resistant pseudomonas aeruginosa
The Journal of Infectious Diseases, 2019Co-Authors: Krisztina M Pappwallace, Marisa L Winkler, Roshan Dsouza, Indresh Singh, Granger G Sutton, Steven Park, Elise T Zeiser, Scott A Becka, Brigid M Wilson, Derrick E FoutsAbstract:Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-Avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-Avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-Avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to β-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-Avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-Avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-Avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-Avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-Avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-β-lactamases.
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overcoming an extremely drug resistant xdr pathogen Avibactam restores susceptibility to ceftazidime for burkholderia cepacia complex isolates from cystic fibrosis patients
ACS Infectious Diseases, 2017Co-Authors: Krisztina M Pappwallace, Elise T Zeiser, Scott A Becka, Nozomi Ohuchi, Maria F. Mojica, Julian A. Gatta, Monica Falleni, Delfina Tosi, Elisa Borghi, Marisa L WinklerAbstract:Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-β-lactam β-lactamase inhibitor, Avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of Avibactam). Furthermore, a major β-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by Avibactam with a high k2/K of (2 ± 1) × 106 μM–1 s–1 and a slow koff of (2 ± 1) × 10–3 s–1. Mass spectrometry revealed that Avibactam formed a stable complex with PenA for up to 24 h and that Avibactam recyclized off of PenA, re-forming the active compound. Crystallograph...
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activity of ceftazidime Avibactam against isogenic strains of escherichia coli containing kpc and shv β lactamases with single amino acid substitutions in the ω loop
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Marisa L Winkler, Krisztina M Pappwallace, Robert A BonomoAbstract:Objectives: The objective of this study was to explore the activity of ceftazidime and ceftazidime/Avibactam against a collection of isogenic strains ofEscherichiacoli DH10B possessing SHV and KPCb-lactamases containing single amino acid substitutions in the V-loop (residues 164 –179). Methods: Ceftazidime and ceftazidime/Avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 b-lactamase variants that possessed elevated MICs of ceftazidime/Avibactam were selected for further biochemical analyses. Results: Avibactam restored susceptibility to ceftazidime for all V-loop variants of SHV-1 with MICs ,8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/Avibactam .8 mg/L. b-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 b-lactamases indicated that Avibactam effectively inhibited these enzymes. Conclusions: Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in theV-loop were not susceptible to ceftazidime/Avibactam (MICs .8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant b-lactamases that prevent Avibactam from binding to and inhibiting the b-lactamase. As ceftazidime/ Avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important.
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activities of ceftazidime ceftaroline and aztreonam alone and combined with Avibactam against isogenic escherichia coli strains expressing selected single β lactamases
Diagnostic Microbiology and Infectious Disease, 2015Co-Authors: Krisztina M Pappwallace, Marisa L Winkler, Robert A Bonomo, Wright W. Nichols, Raymond T Testa, Julian A. Gatta, Saralee Bajaksouzian, Ayman M Abdelhamed, Altreisha N Foster, Michael R JacobsAbstract:Abstract Avibactam is a novel β-lactamase inhibitor that restores the activity of otherwise hydrolyzed β-lactams against Gram-negative bacteria expressing different classes of serine β-lactamases. In the last decade, β-lactam–Avibactam combinations were tested against a variety of clinical isolates expressing multiple commonly encountered β-lactamases. Here, we analyzed isogenic Escherichia coli strains expressing selected single β-lactamase genes that were not previously tested or were not characterized in an isogenic background. The activities of ceftazidime, ceftaroline, and aztreonam alone and in combination with 4mg/L of Avibactam, as well as comparator agents, were assessed against a unique collection of isogenic strains of E. coli carrying selected extended-spectrum, inhibitor-resistant, and/or carbapenem-hydrolyzing bla genes. When combined with Avibactam, ceftazidime, ceftaroline, or aztreonam MICs were reduced for 91.4%, 80.0%, and 80.0% of isolates, respectively. The data presented add to our understanding of the microbiologic spectrum of these β-lactams with Avibactam and serve as a reference for further studies.
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ceftazidime Avibactam in combination with fosfomycin a novel therapeutic strategy against multidrug resistant pseudomonas aeruginosa
The Journal of Infectious Diseases, 2019Co-Authors: Krisztina M Pappwallace, Marisa L Winkler, Roshan Dsouza, Indresh Singh, Granger G Sutton, Steven Park, Elise T Zeiser, Scott A Becka, Brigid M Wilson, Derrick E FoutsAbstract:Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-Avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-Avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-Avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to β-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-Avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-Avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-Avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-Avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-Avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-β-lactamases.
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overcoming an extremely drug resistant xdr pathogen Avibactam restores susceptibility to ceftazidime for burkholderia cepacia complex isolates from cystic fibrosis patients
ACS Infectious Diseases, 2017Co-Authors: Krisztina M Pappwallace, Elise T Zeiser, Scott A Becka, Nozomi Ohuchi, Maria F. Mojica, Julian A. Gatta, Monica Falleni, Delfina Tosi, Elisa Borghi, Marisa L WinklerAbstract:Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-β-lactam β-lactamase inhibitor, Avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of Avibactam). Furthermore, a major β-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by Avibactam with a high k2/K of (2 ± 1) × 106 μM–1 s–1 and a slow koff of (2 ± 1) × 10–3 s–1. Mass spectrometry revealed that Avibactam formed a stable complex with PenA for up to 24 h and that Avibactam recyclized off of PenA, re-forming the active compound. Crystallograph...
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Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients
2017Co-Authors: Krisztina M. Papp-wallace, Elise T Zeiser, Scott A Becka, Nozomi Ohuchi, Maria F. Mojica, Julian A. Gatta, Monica Falleni, Delfina Tosi, Elisa Borghi, Marisa L WinklerAbstract:Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-β-lactam β-lactamase inhibitor, Avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of Avibactam). Furthermore, a major β-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by Avibactam with a high k2/K of (2 ± 1) × 106 μM–1 s–1 and a slow koff of (2 ± 1) × 10–3 s–1. Mass spectrometry revealed that Avibactam formed a stable complex with PenA for up to 24 h and that Avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA–Avibactam revealed several interactions that stabilized the acyl–enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime–Avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime–Avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime–Avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials
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activity of ceftazidime Avibactam against isogenic strains of escherichia coli containing kpc and shv β lactamases with single amino acid substitutions in the ω loop
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Marisa L Winkler, Krisztina M Pappwallace, Robert A BonomoAbstract:Objectives: The objective of this study was to explore the activity of ceftazidime and ceftazidime/Avibactam against a collection of isogenic strains ofEscherichiacoli DH10B possessing SHV and KPCb-lactamases containing single amino acid substitutions in the V-loop (residues 164 –179). Methods: Ceftazidime and ceftazidime/Avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 b-lactamase variants that possessed elevated MICs of ceftazidime/Avibactam were selected for further biochemical analyses. Results: Avibactam restored susceptibility to ceftazidime for all V-loop variants of SHV-1 with MICs ,8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/Avibactam .8 mg/L. b-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 b-lactamases indicated that Avibactam effectively inhibited these enzymes. Conclusions: Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in theV-loop were not susceptible to ceftazidime/Avibactam (MICs .8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant b-lactamases that prevent Avibactam from binding to and inhibiting the b-lactamase. As ceftazidime/ Avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important.
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activities of ceftazidime ceftaroline and aztreonam alone and combined with Avibactam against isogenic escherichia coli strains expressing selected single β lactamases
Diagnostic Microbiology and Infectious Disease, 2015Co-Authors: Krisztina M Pappwallace, Marisa L Winkler, Robert A Bonomo, Wright W. Nichols, Raymond T Testa, Julian A. Gatta, Saralee Bajaksouzian, Ayman M Abdelhamed, Altreisha N Foster, Michael R JacobsAbstract:Abstract Avibactam is a novel β-lactamase inhibitor that restores the activity of otherwise hydrolyzed β-lactams against Gram-negative bacteria expressing different classes of serine β-lactamases. In the last decade, β-lactam–Avibactam combinations were tested against a variety of clinical isolates expressing multiple commonly encountered β-lactamases. Here, we analyzed isogenic Escherichia coli strains expressing selected single β-lactamase genes that were not previously tested or were not characterized in an isogenic background. The activities of ceftazidime, ceftaroline, and aztreonam alone and in combination with 4mg/L of Avibactam, as well as comparator agents, were assessed against a unique collection of isogenic strains of E. coli carrying selected extended-spectrum, inhibitor-resistant, and/or carbapenem-hydrolyzing bla genes. When combined with Avibactam, ceftazidime, ceftaroline, or aztreonam MICs were reduced for 91.4%, 80.0%, and 80.0% of isolates, respectively. The data presented add to our understanding of the microbiologic spectrum of these β-lactams with Avibactam and serve as a reference for further studies.
