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Home , Avibactam, Piperacillin, Sulbactam, Tazobactam, Ticarcillin

Susceptibility testing β-lactamase inhibitor combinations

Derek Brown Scientific Secretary EUCAST

22 June 2016, BSAC user day β-lactamase inhibitor combinations

• Active area of development of antimicrobial agents

• Questions about changing from 2:1 ratio to fixed inhibitor concentration for -

• Concerns about the reliability of susceptibility testing β-lactamase inhibitors

• Clavulanic acid (1976)

• (1980)

• (1984)

• (2009)

β-lactamase inhibitor combinations

Inhibitor Combined with

Clavulanic acid Amoxicillin

Sulbactam Tazobactam Ceftolozane Avibactam Fixed concentration of inhibitor or ratio with active agent for in vitro tests?

• Amoxicillin-clavulanic acid historically tested with 2:1 ratio

• ampicillin-sulbactam also 2:1 ratio “for comparability with amoxicillin-clavulanic acid”

• All later combinations tested with fixed concentration of inhibitor Why amoxicillin:clavulanic acid originally in 2:1 ratio for in vitro tests?

• Amoxicillin:clavulanic acid in 2:1 ratio in pharmaceutical preparations.

• Pharmacology similar so ratio maintained in patients

• “Optimal” activity in in vitro tests

• Promoted by manufacturer and widely accepted

but………. Why not amoxicillin:clavulanic acid in 2:1 ratio for in vitro tests? • Amoxicillin:clavulanic acid ratio in pharmaceutical preparations varies widely, from 2:1 to 16:1 (oral) and 5:1 to 10:1 (iv)

• Pharmacology similar but some differences so same ratio is not maintained at all sites of infection

• At higher amoxicillin MICs the concentration of clavulanic acid is higher than achieved clinically and will have an antibacterial effect. At lower amoxicillin MICs the concentration of clavulanic acid will be lower than achieved clinically Why not amoxicillin:clavulanic acid in 2:1 ratio for in vitro tests?

• The objective is to assess whether the β- lactamase inhibitor (clavulanic acid) will affect the MIC of the active agent (amoxicillin), so a fixed concentration of inhibitor is more logical

• PK-PD studies suggest that fixed concentrations of inhibitors are appropriate Ratio or fixed concentration for in vitro tests?

• Argument for a fixed concentration of inhibitor now widely accepted

• EUCAST breakpoints are based on fixed inhibitor concentration for amoxicillin-clavulanic acid and amoxicillin-sulbactam, as for all other β-lactamase inhibitor combinations Issues with changing from amoxicillin:clavulanic acid in 2:1 ratio to a fixed 2 mg/L clavulanic acid for MIC tests

• Amoxicillin-clavulanic acid appears less active

• Resistance rates suddenly increased

• Problems with resistance surveillance data Amoxicillin-clavulanic acid MICs for E. coli with clavulanic acid fixed 2 mg/L (n=10635) or amox:clav 2:1 ratio (n=22094). EUCAST aggregated data 40 fixed 35 2:1ratio

30

25

20

15 Percent isolates Percent 10

5

0 ≤1 2 4 8 16 32 64 ≥128 MIC (mg/L) Amoxicillin-clavulanic acid MICs for Enterobacteriaceae with clavulanic acid fixed 2 mg/L or amox:clav 2:1 ratio Selected isolates tested in parallel (n=60) 60

Fixed 2 50

2:1 ratio

40

30

20 Number of of isolates Number

10

0 ≤1 2 4 8 16 32 64 ≥128 MIC (mg/L) Concentration of inhibitor for MIC determination?

• The concentration of inhibitor needed to inhibit the • Any intrinsic activity of the inhibitor • PK-PD for the inhibitor • Clinical studies relating outcome to in vitro tests Concentration of clavulanic acid required to inhibit β-lactamases

• TEM-1 requires 5.4-12.7 mg/L clavulanic acid to reduce the amoxicillin MIC to 8 mg/L (Livermore. JAC 1993; 31 suppl A:9-21).

• MICs of 25% isolates with TEM-1 reduced to ≤8 mg/L by 2 mg/L clavulanic acid and 65% isolates by 4 mg/L clavulanic acid (Thomson et al AAC 1995; 39: 2591- 2592). Concentration of clavulanic acid required to inhibit β-lactamases • Depends on the particular β-lactamase and the amount of β-lactamase produced Ampicillin MIC (mg/L) with 8 mg/L clavulanic acid for E. coli with different β-lactamases TEM-1 8 SHV-1 16 TEM-2 64 SHV-2 16 TEM-3 8 SHV-3 16 TEM-4 8 SHV-4 8 TEM-5 8 SHV-5 8 TEM-6 16 TEM-7 16 TEM-9 8

Intrinsic activity of the inhibitor (from Bryskier, Antimicrobial Agents, ASM 2005)

Organisms MIC (mg/L) Clavulanic Sulbactam Tazobactam acid Enterobacteriaceae 16-128 32->128 >128 P. aeruginosa >128 >128 >128 spp. 4-16 0.5-2 4-32 S. aureus 16 32->128 32 Haemophilus spp. 32 128 64 Neisseria spp. 0.03-8 0.03-2 PK-PD for β-lactamase inhibitor combinations

• Models being developed for newer inhibitors • Need to identify PK-PD targets for inhibitor and active agent and apply both in model

• Targets for different inhibitors may be different, e.g. • For tazobactam target of 60% fT>0.5 x ceftolozane-tazobactam MIC (Ambrose) • For avibactam target of 50% fT>2.5 mg/L (Ambrose) • For avibactam target of 60% fT>1 mg/L (Mouton)

• No data on amoxicillin-clavulanic acid

Pharmacokinetics of clavulanic acid

• Amount of clavulanic acid in single dose varies depending on the preparation. • Up to 200 mg in iv preparations. – peak concentration in serum around 12-16 mg/L, with a half life of 0.8-1h and 25-30% protein binding – Approx. 2 mg/L maintained for half dosing interval • Up to 125 mg in oral preparations – peak concentration in serum around 2.5 mg/L – 50% eliminated via urine

Clinical studies relating outcome to in vitro tests for different concentrations of inhibitor

None

MIC breakpoints for β-lactamase inhibitor combinations • Use a fixed concentration of inhibitor – Clavulanic acid 2 mg/L – Sulbactam 4 mg/L – Tazobactam 4 mg/L – Avibactam 4 mg/L • Breakpoint for active agent same as the agent without the β-lactamase inhibitor e.g. amoxicillin- clavulanic acid – Enterobacteriaceae 8 mg/L – Enterococcus spp. 8 mg/L – 2 mg/L – Moraxella catarrhalis 1 mg/L

MIC determination for β-lactamase inhibitor combinations • Broth microdilution - fixed concentration of inhibitor • Agar dilution - fixed concentration of inhibitor

• Gradient tests - limited availability of fixed concentration Agent bioMérieux Liofilchem ThermoFisher Etest MIC test strip M.I.C. evaluator Amoxicillin-clavulanic acid No Yes No Ampicillin-sulbactam No Yes No Ticarcillin-clavulanic acid Yes Yes No Piperacillin-tazobactam Yes Yes No MIC distributions for β-lactamase inhibitor combinations MIC distributions for β-lactamase inhibitor combinations MIC distributions for β-lactamase inhibitor combinations Disk diffusion susceptibility tests for β-lactamase inhibitor combinations • Fixed concentration of inhibitor not possible • Ratio in disks – 20-10 µg or 2-1 µg amoxicillin-clavulanic acid – 10-10 µg ampicillin-sulbactam – 30-6 µg piperacillin-tazobactam – 75-10 µg ticarcillin-clavulanic acid • Calibration against MIC with fixed concentration of inhibitor – Different amounts of active agents and inhibitor – Different media manufacturers – Different disk manufacturers

Amoxicillin-clavulanic acid (fixed 2 mg/L) MIC v zone diameter for Enterobacteriaceae by EUCAST method E. coli, Citrobacter spp., Klebsiella spp., spp., Serratia spp., Proteus spp., M. morganii

≥128 25 1 6 3 1 1 4 5 4 1 1 1 1

64 1 1 1

32 1 1 1

16 1 5 2 1 1

8 2 2 1 2

MIC (mg/L) MIC 4 1 1 1 1 2

2 1 2 2 1

1 1 2 1 2

≤0.5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Zone diameter (mm)

Data for NEQAS/BSAC/EUCAST study 2011-12 Amoxicillin-clavulanic acid 20-10 µg vs. MIC E. coli, 133 clinical isolates 18 (3 data sources)

16 • Breakpoints for uncomplicated UTI. • Fixed concentration of clavulanic acid MIC at 2 mg/L. 14 (mg/L) ≥128 12 64 10 32 8 16

8 No of isolates of No 6 4 2 4 1 2

0

6 8

34 10 12 14 16 18 20 22 24 26 28 30 32 36 38 40 Inhibition zone diameter (mm)

Breakpoints (uncomplicated UTI) ECOFF MIC S≤32, R>32 mg/L 8 mg/L Zone diameter S≥16, R<16 mm Amoxicillin-clavulanic acid 20-10 µg vs. MIC E. coli, 133 clinical isolates 18 (3 data sources)

16 • Breakpoints for systemic infections. • Fixed concentration of clavulanic acid MIC at 2 mg/L. 14 (mg/L) ≥128 12 64 10 32 8 16

8 No of isolates of No 6 4 2 4 1 2

0

6 8

34 10 12 14 16 18 20 22 24 26 28 30 32 36 38 40 Inhibition zone diameter (mm)

Breakpoints (systemic infections) ECOFF MIC S≤8, R>8 mg/L 8 mg/L Zone diameter S≥19, R<19 mm Piperacillin-tazobactam 30-6 µg vs. MIC aeruginosa, 249 isolates (356 correlates) 60 (7 data sources)

MIC 50 (mg/L)

≥128 40 64 32 30 16 8 4 20 No of observations of No 2 1 10 ≤0.5

0

6 8

34 10 12 14 16 18 20 22 24 26 28 30 32 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤16, R>16 mg/L 16 mg/L Zone diameter S≥18, R<18 mm Automated susceptibility tests for β-lactamase inhibitor combinations

• Broth based so fixed concentration of inhibitor – Clavulanic acid fixed concentration for amoxicillin- clavulanic acid not available in Vitek and Microscan – Sulbactam fixed concentration in ampicillin-sulbactam not available in any system • Performance should be in line with MIC tests • 2007-8 reports of failure of Vitek to detect ESBL- mediated resistance to piperacillin-tazobactam

Control of β-lactam-β-lactamase inhibitor combinations • β-lactamase inhibitors, particularly clavulanic acid, are not stable • Quality control of inhibitor content of combined disks and inhibitors in MIC tests on inhibitor combinations should include a control for activity of the inhibitor ……obvious but rarely done Control of β-lactam-β-lactamase inhibitor combinations • The inhibitor component should be controlled with E. coli ATCC 35218 (TEM-1 β-lactamase-producing strain) – Should be part of routine QC

• E. coli ATCC 25922 is used to control the active component

Control of β-lactam-β-lactamase inhibitor combinations

EUCAST QC table v 5.0 ATCC 35218 (NCTC 11954, CIP 102181, DSM 5923, CCUG 30600, CECT 943) TEM-1 β-lactamase-producing strain (non-ESBL) used to check the inhibitor component of inhibitpr combination disks.

Disk diffusion methodology: Mueller-Hinton agar, McFarland 0.5, air, 35±1ºC, 18±2h. Read zone edges as the point showing no growth viewed from the back of the plate against a dark background illuminated with reflected light.

MIC Inhibition zone diameter Disk content Antimicrobial agent (mg/L) (mm) (µg) Target1 Range2 Target1 Range2 Amoxicillin-clavulanic acid3 8-16 4-32 20-10 20 17-224 Ampicillin-sulbactam5 32-64 16-128 10-10 16 13-194 Piperacillin-tazobactam6 1 0.5-2 30-6 24 21-27 Ticarcillin-clavulanic acid3 16 8-32 75-10 23 21-25 3 The concentration of clavulanic acid is fixed at 2 mg/L. 5 The concentration of sulbactam is fixed at 4 mg/L. 6 The concentration of tazobactam is fixed at 4 mg/L. Performance of UKNEQAS participants in susceptibility testing of inhibitor combinations

MIC Number Organisms Agent Concordance (mg/L) of isolates Enterobacteriaceae Amoxicillin- ≤8 5 ≥94% S 2014-16 clavulanic ≥128 10 ≥95% R acid Piperacillin- ≤8 6 ≥98% S tazobactam ≥128 4 ≥99% R 1 67% R 16-64 6 27-94% R P. aeruginosa Piperacillin- ≤16 8 ≥97% S 2012-16 tazobactam 1 72% S 32-64 3 ≥91% R 1 43% R Performance of UKNEQAS participants in susceptibility testing of E. coli 2832 against piperacillin-tazobactam (MIC ≥128 mg/L, plasmid AmpC, BIL-1)

Number (%) participants Guideline Method n reporting S I R Automated 248 1.6 36.7 61.7 EUCAST Disk diffusion 170 12.9 5.3 81.8 MIC 40 2.5 22.5 75.0 Automated 29 13.8 51.7 34.5 CLSI Disk diffusion 26 38.5 42.3 19.2 MIC 11 27.3 0 72.7 Performance of UKNEQAS participants in susceptibility testing of E. coli 2253 against piperacillin-tazobactam (MIC 32-64 mg/L, CTX-M-15)

Number (%) participants Guideline Method n reporting S I R Automated 208 53.4 12.5 31.4 EUCAST Disk diffusion 235 27.6 29.4 43.0 MIC Automated 34 70.6 23.5 5.9 CLSI Disk diffusion 30 63.3 20.0 16.7 MIC Conclusions β-lactamase inhibitor combinations

• MIC breakpoints based on tests with fixed inhibitor concentration

• Fixed inhibitor concentration for amoxicillin- clavulanic acid not implemented in some commercial systems

• EUCAST disk diffusion method calibrated to fixed inhibitor concentrations

• Concerns about the reliability of susceptibility testing when MICs are close to breakpoints