Susceptibility testing new agents

.

Dr. Ian Morrissey Chief Executive GR Micro Ltd. London, UK.

New Agents

• Linezolid (Pfizer) • Tigecycline (Wyeth) • Daptomycin (Novartis) • Dalbavancin (Pfizer)

Basic principles still important.

Linezolid

• Approved in 2000 in the USA.

• Community-acquired and nosocomial pneumonia.

• Complicated and uncomplicated skin and soft-tissue infections.

• Methicillin-resistant Staphylococcus aureus resistant enterococci. and vancomycin • Protein synthesis inhibitor • Bacteriostatic agent • Oral and parenteral formulations.

Linezolid susceptibility

• Non-susceptibility is rare – Some development signs in Enterococci esp. E. faecium .

– Occasional cases in S. aureus .

– Very rare in S. pneumoniae .

Linezolid MIC Breakpoints mg/L (≤S/>R) Staphylococcus Enterococcus Streptococcus A,B,C,G

S.pneumoniae

Non-species related 4/4 4/4 4/4 2/4 2/4 Zone diameters, 10 µg disc (mm) ≥S I ≤R 19 19 20 20 19 19 20 20 -

CLSI modified guidelines

• QC ranges changed from 27-31 mm to 25-32 mm after 4-5 yrs of use.

• Bacteriostatic agents may be more problematical when measuring zones or MICs by Etest.

Biedenbach & Jones 2003 CMI 9:1035

Tigecycline

• Launched 2005 in USA for SSTI.

• First glycylcycline – Derivative of minocycline – Evades Tet A-E & K efflux pumps – But not those of Proteae or Pseudomonas • Unique amongst newer agents having activity against Gram negative bacteria.

– But is active against MRSA and VRE.

– Acinetobacter baumannii • Parenteral administration only.

• Bacteriostatic.

Tigecycline susceptibility

• High against Gram-positive bacteria, E. coli & K. oxytoca .

• Some resistance found in Gram-negs – – K. pneumoniae (92-95% susceptibility) E. aerogenes (96% susceptibility) – – E. cloacae (93% susceptibility) S. marcescens (97% susceptibility)

Waites et al 2006 AAC 50: 3479

Tigecycline breakpoints

Tigecycline MIC Breakpoints mg/L (≤S/>R) Enterobacteriaceae Staphylococcus Enterococcus Streptococcus Non-species related 1/2 0.5/0.5

0.25/0.5

0.25/0.5

0.25/0.5

Zone diameters, 15 µg disc (mm) ≥S I ≤R 19 20-23 24 25 20 19 26 21 20 -

Medium batch & Tigecycline activity

• Inconsistencies were noticed in QC data.

– Linked to medium batch variation.

• Tetracyclines are prone to inactivation by oxidation.

• Studies carried out to investigate.

– – Petersen & Bradford 2005 AAC 49:3910 Bradford et al 2005 AAC 49:3903.

Old vs new

Resolved by addition of Oxyrase

Chromatographic evidence

H 2 O H 2 O & Oxyrase Aged MHB

Medium effect

• Important for agar dilution or broth dilution.

– Media >12h old affected.

• Does not effect disc diffusion or Etest methods.

• If using broth microdilution frozen panels are also not affected.

• Solved by boiling or addition of oxyrase.

Daptomycin

• First glycolipopeptide • Launched in 2003 in the USA – 2006 in Europe – SSTI – Endocarditis in the USA • Gram-positives only • Parenteral application only • Rapidly bactericidal

Daptomycin Susceptibility

• Non-susceptibility rare.

– Some reports but case studies only.

Daptomycin MIC Breakpoints mg/L (≤S/>R) Staphylococcus

[Enterococcus

Streptococcus 1/1

4/4]

1/1

[CLSI only]

≥S Zone diameters (mm) I ≤R -

-

-

-

-

Daptomycin – requires Calcium

Calcium effect

MIC at zero Ca++ = 64 mg/L GR Micro Ltd, data on file.

Daptomycin diffusion assays

• Daptomycin discs supplemented with 50 mg/L Ca++ have been developed.

– Only for CLSI methodology (i.e. MHB) – Discontinued in 2005 • Isosensitest discs have proved problemmatical and have never been available.

Why are dapto & Ca++ discs no longer available even for CLSI?

Daptomycin treatment failure

Susceptible BP was ≥16 mm

Hayden et al 2005 JCM 43:5285.

Multicentre evaluation

Jevitt et al 2006 JCM 44:3098

Etest to the rescue!

Jevitt et al 2006 JCM 44:3098

Dalbavancin

• New lipoglycopeptide – Derived from teicoplanin • Extended half-life – Once weekly dosing • Bactericidal • 8-16-fold more active than vancomycin • No resistance found to date – Except for cross-resistance to vanA Enterococci.

• Not clinically available but has ‘FDA Approval’

Dalbavancin Susceptibility

• Breakpoints not currently set.

• Broth microdilution requires addition of polysorbate-80 into wells for accurate and reproducible results.

• Agar diluton has not been proposed as a standard method – Fritsche et al 2006 JCM 44:2988 • Problems with disc development – Poor agar diffusion – Jones et al 2006 JCM 44:2622 • Etest is available.

– Correct interpretation is essential.

Dalbavancin Etest

Biedenbach et al 2007 JCM 45:998

BSAC agar dilution method

• BSAC method compares well with CLSI (NCCLS) – Mustaq et al 2004 JAC 54:617.

• Agar dilution commonly 1 dilution higher

Surrogate marker for dalbavancin

Jones et al 2006 JCM 44:2622

Summary

• Linezolid – Continued use has ironed-out previous QC issues • Tigecycline – For broth: fresh medium or oxyrase is essential • Daptomycin – Discs not available – but Etests work.

– Ca++ supplement required • Dalbavancin – Some question over agar dilution but BSAC seems OK.

– Polysorbate-80 supplement in broth.

– No disc but Etest is available.