Susceptibility testing new agents.

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Susceptibility testing new agents.

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

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New Agents

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

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Basic principles still important.
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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.

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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/gtR) Linezolid MIC Breakpoints mg/L (S/gtR) Zone diameters, 10 µg disc (mm) Zone diameters, 10 µg disc (mm) Zone diameters, 10 µg disc (mm)
Linezolid MIC Breakpoints mg/L (S/gtR) Linezolid MIC Breakpoints mg/L (S/gtR) 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  - - -
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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 91035
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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.

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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
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Tigecycline breakpoints
Tigecycline MIC Breakpoints Tigecycline MIC Breakpoints Zone diameters, 15 µg disc (mm) Zone diameters, 15 µg disc (mm) Zone diameters, 15 µg disc (mm)
mg/L (S/gtR) mg/L (S/gtR) 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 - - -
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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 493910
• Bradford et al 2005 AAC 493903.

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Old vs new
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Resolved by addition of Oxyrase
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Chromatographic evidence
H2O
H2O Oxyrase
Aged MHB
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Medium effect

• Important for agar dilution or broth dilution.
• Media gt12h 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.

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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

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Daptomycin Susceptibility

• Non-susceptibility rare.
• Some reports but case studies only.

Daptomycin MIC Breakpoints Daptomycin MIC Breakpoints Zone diameters (mm) Zone diameters (mm) Zone diameters (mm)
mg/L (S/gtR) mg/L (S/gtR) S I R
Staphylococcus 1/1 - - -
Enterococcus 4/4 - - -
Streptococcus 1/1 - - -
CLSI only
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Daptomycin requires Calcium
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Calcium effect
MIC at zero Ca 64 mg/L
GR Micro Ltd, data on file.
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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?

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Daptomycin treatment failure
Susceptible BP was 16 mm
Hayden et al 2005 JCM 435285.
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Multicentre evaluation
Jevitt et al 2006 JCM 443098
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Etest to the rescue!
Jevitt et al 2006 JCM 443098
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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

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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 442988
• Problems with disc development
• Poor agar diffusion
• Jones et al 2006 JCM 442622
• Etest is available.
• Correct interpretation is essential.

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Dalbavancin Etest
Biedenbach et al 2007 JCM 45998
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BSAC agar dilution method

• BSAC method compares well with CLSI (NCCLS)
• Mustaq et al 2004 JAC 54617.
• Agar dilution commonly 1 dilution higher

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Surrogate marker for dalbavancin
Jones et al 2006 JCM 442622
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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.