Susceptibility testing new agents.

Dr. Ian MorrisseyChief ExecutiveGR 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 and

vancomycin-resistant enterococci. • 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 Breakpointsmg/L (≤S/>R)

Zone diameters, 10 µg disc (mm)

≥S I ≤R

Staphylococcus 4/4  19 - 20

Enterococcus 4/4  19 - 20

Streptococcus A,B,C,G 4/4  19 - 20

S.pneumoniae 2/4  19 - 20

Non-species related 2/4  - - -

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 Zone diameters, 15 µg disc (mm)

mg/L (≤S/>R) ≥S I ≤R

Enterobacteriaceae 1/2 19 20-23 24

Staphylococcus 0.5/0.5 25 - 26

Enterococcus 0.25/0.5 20 - 21

Streptococcus 0.25/0.5 19 - 20

Non-species related 0.25/0.5 - - -

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

H2O

H2O & 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 Zone diameters (mm)

mg/L (≤S/>R) ≥S I ≤R

Staphylococcus 1/1 - - -

[Enterococcus 4/4] - - -

Streptococcus 1/1 - - -

[CLSI only]

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

Hayden et al 2005 JCM 43:5285.

Susceptible BP was ≥16 mm

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.