Mechanisms of Resistance to Ceftazidime-Avibactam Romney M. Humphries, PhD D(ABMM) Chief Scientific Officer Accelerate Diagnostics
UCLA, January 2015 62 year old woman with advanced pancreatic cancer Vomiting & fever after surgery CT scan: fluid collection in liver, inflammatory ascites Blood cultures: Gram negative rods ID: Klebsiella pneumoniae R to all drugs tested Disk diffusion test results for our patient s K. pneumoniae Would the new drug, ceftazidime- avibactam, have activity against this isolate? Humphries et al. Antimicrob. Agents Chemother. 2015 vol. 59 6605-7
Ceftazidime-avibactam (CZA) in 2015 Activity against serine beta-lactamases, including KPC Isolate tested and found to have KPC-3 No activity vs. class B beta-lactamases (e.g., NDM) Isolate negative for MBLs (by PCR and by WGS) So should work, right? 19 mm = Resistant* * MIC confirmed resistance, 16 32 ug/ml First case of KPC-producer resistant to CZA Isolated 1 month prior to CZA FDA-approval Antimicrob. Agents Chemother. 2017 vol. 61 e00537-17
Ceftazidime-avibactam Avibactam is a first in class diazabicyclooctane beta-lactamase inhibitor Reversible covalent binding to beta-lactamase enzyme Inhibits activity of: Class A beta-lactamases ESBL, KPC Class C beta-lactamases Some class D beta-lactamases Inactive against Class B beta-lactamases weak binding to this class of enzymes Indications: ciai (with metronidazole) cuti HAP Treatment of GN with limited options (EU)
Clinical breakpoints for CZA CLSI / FDA Organism Group S I R Enterobacteriaceae MIC Disk (30/20) P. aeruginosa MIC Disk (30/20) 8/4 µg/ml 21 mm 8/4 µg/ml 21 mm - 16/4 µg/ml 20 mm - 16/4 µg/ml 20 mm EUCAST Organism Group S I R Enterobacteriales MIC Disk (10/4) P. aeruginosa MIC Disk (10/4) 8/4 mg/l 13 mm 8/4 mg/l 17 mm - >8 mg/l <13 mm - >8 mg/l <17 mm
Clinical breakpoints for CZA CLSI / FDA Organism Group S I R Enterobacteriaceae MIC Disk (30/20) P. aeruginosa MIC Disk (30/20) 8/4 µg/ml 21 mm 8/4 µg/ml 21 mm - 16/4 µg/ml 20 mm - 16/4 µg/ml 20 mm EUCAST Organism Group S I R Enterobacteriales MIC Disk (10/4) P. aeruginosa MIC Disk (10/4) 8/4 mg/l 13 mm 8/4 mg/l 17 mm - >8 mg/l <13 mm - >8 mg/l <17 mm
Reports of CZA resistance Study performed at MD Anderson, Texas Unexpectedly high rate of endemic NDM-1 (not outbreak) 64% of CRE were NDM, all CZA-R UCLA data: sporadic cases, 4-5 per year
KPC-2 vs. KPC-3 CZA MICs Shields et al. 2015. AAC 59: 579
The Omega Loop Conserved motif in class A beta-lactamases Arg164 Asp179 Glu166 and Asn170: de-acylation of beta-lactam AA substitution here = disruption of R164-D179 salt bridge = flexible increased hydrolytic activity Winkler ML, Papp-Wallace KM and Bonomo RA. 2015. JAC 70:2279
In vitro: mutagenesis of omega loop Mutation Ceftazidime MIC CAZ-AVI MIC KPC-2 64 1 Arg164Ala 256 16 Arg164His 256 8 Arg164Pro 256 64 Arg164Ser 256 8 Leu167Ala 32 1 Leu167Pro 16 1 Leu167Thr 32 1 Leu169Ala 128 2 Leu169Arg 256 0.5 Asp179Ala 512 64 Winkler ML, Papp-Wallace KM and Bonomo RA. 2015. JAC 70:2279 Evaluated R164A and D179N Avibactam kinetics: Similar to KPC-2 Ceftazidime kinetics: Rapid binding Mutation to omega loop: increased affinity for CAZ, prevents binding of AVI
Activity of other beta-lactam combinations with avibactam unaffected by Asp179 mutation Strain CAZ CAZ- AVI CRO AZT TAR TOL- TAZ TOL- TAZ- AVI CRO- AVI AZT- AVI TAR- AVI KPC-2 128 0.25 32 1 64 0.125 256 0.25 256 0.25 D179N 512 16 16 8 16 0.5 256 0.5 128 1.0 D179Y >512 64 16 16 16 1.0 8 0.5 32 2.0 * Meropenem activity diminished in all Barnes et al. 2017. mbio. 8:e00528-17
Mutation to D179N D179N vs. KPC-2: rate of CAZ, IMI hydrolysis decreased rate of deacylation affinity for CAZ and other beta-lactams flexible / mobile Disrupted salt bridge between D179 - R164 Change to hydrogen bond network with other AAs Altered position of catalytic S70 Enlarged active site Barnes et al. 2017. mbio. 8:e00528-17 D179N traps ceftazidime
Omega Loop: In the clinic (KPC-3) 10 / 37 patients failed therapy (microbiological recurrence) 3 of these had CZA R isolates CAZ (ug/ml) CZA (ug/ml) MER (ug/ml) WT 256 0.5 4 D179Y 256 8 0.06 T243M 64 8 0.125 D179Y/T243M 512 16 0.06 EL165-166 128 4 0.125 Haidar G et al. 2017 AAC 61:E02534-16
Omega Loop: In the clinic (KPC-3) Isolate 4-A recovered from respiratory secretions (VAP), rx with 1.25 g q8 CAZ-AVI Isolate 4-C from blood, treated with meropenem 1g q12 for 18 days Circle, no drug Square = 16 ug/ml Cross = 8 x MIC Triangle = 4 x MIC
MIC, ug/ml Serial passage in meropenem reverses phenotype >64 128 64 32 16 8 4 2 1 0.5 0.25 Base Week 1 Week 2 Week 3 Week 4 Week 5 At end of passage: - A177E or T243A - reversion to KPC-3 Most retained CZA resistance OmpK36 mutations also observed V240G T243A D179Y A177E, D179Y D179Y, T243M
Omega Loop: In the clinic (KPC-2) UCLA, patient with cirrhosis, sepsis CRE isolated from BAL (VAP), rx with ceftazidime-avibactam 1 month later, BSI with ESBL K. pneumoniae rx with meropenem (later found to be KPC-2 producer) KPC-2 L169P mutation, Ceftazidime-avibactam MIC = 16 ug/ml Strain Ceftriaxone Ceftazidime Ceftolozanetazobactam Cefepime Imipenem Meropenem CAZ/AVI DH5a <0.5 <0.5 <0.5 <0.5 <0.25 <0.25 0.25 DH5a, KPC2 >32 32 32 >32 >16 >16 0.25 DH5a, L169P 4 >32 >32 2 <0.25 <0.25 4 Humphries and Hemarajata, in preparation
Beyond the omega loop: decreased permeability and increased hydrolysis UCLA patient: KP1244: KPC-3, WT Ceftazidime-avibactam: 32 ug/ml Nelson et al. 2017 AAC. 61:e00989 - Increased KPC-3 &SHV-12 (not shown) expression - Increased CAZ and MEM hydrolysis - Increased copy number of blakpc (log phase)
Beyond the omega loop: decreased permeability and increased hydrolysis Decreased permeability: Nonfunctional OmpK35 (truncated at position 42) Mutations to OmpK36 = R191L and T333N Decreased uptake of meropenem demonstrated Nelson et al. 2017 AAC. 61:e00989 Efflux Increased hydrolysis Decrease permeability
CZA resistance: testing issues Challenges with CZA testing: Few commercial methods (automated systems) Reflex testing only for CRE will KPC mutants be found? Differences in disk content CLSI vs. EUCAST Absence of intermediate category
Disk testing issues Disk in US and EU differ (30/20 vs. 10/4) Data used to generate disk breakpoints in US:
Testing issues: Disk (US) Wenzler et al. Submitted Red: P. aeruginosa Black: Enterobacteriales Grey: Major errors (25%)
Testing issues: Disk (EU) Red: P. aeruginosa Black: Enterobacteriales Grey: Major errors (16.3%)
Absence of I for MIC MIC distribution of P. aeruginsa resistant to >1 beta-lactam Isolates with bla VIM had MICs 8 16 ug/ml (S or R) Humphries et al 2017. AAC. 22: e01858-17
38 patients with CZA 99 patients with colistin 46% BSI 22% respiratory IPTW: inverse probability of treatment weighting (adjustment for patient confounders) IPTW-adjusted 30 day all-cause mortality: 9% vs. 32% IPTW adjusted probability of better outcome at 30 days with CZA: 64%
MBL catalysis of avibactam Very slow hydrolysis Aztreonam K m 9 mm; K cat 0.014 s 1 Avibactam K m 3 mm; K cat 0.02 s 1 comparable to: class D β-lactamases vs. carbapenems class C vs. cephalosporins Opportunity for evolution to more efficient catalysis?
Summary Ceftazidime-avibactam resistance: Mutation to KPC-2 or KPC-3 omega loop Increased affinity for ceftazidime, increased susceptibility to meropenem In the clinic: demonstrated in patients post-therapy (microbiological failures) Combination of KPC expression, reduced permeability In the clinic: demonstrated in patient with no exposure Presence of a metallo-beta-lactamase In the clinic: demonstrated to be endemic in some areas, unknown Testing is a challenge: Absence of I category; disk diffusion challenges Nonetheless, CZA is a preferred therapy to traditional options (colistin combination therapy)
Acknowledgements Peera Hemarajata, UCLA Shaun Yang, UCLA / Tricore Aric Gregson, UCLA Paul Allyn, UCLA Olga Lomovskaya, Medicines Company Eric Wenzler, University of Illinois Chicago Funding: Allergan Merck
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