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 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 Breakpoints mg/L (≤S/>R) Zone diameters, 10 µg disc (mm) ≥SI≤R Staphylococcus4/ Enterococcus4/ Streptococcus A,B,C,G4/ S.pneumoniae2/ Non-species related2/4 ---

CLSI modified guidelines QC ranges changed from mm to 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 BreakpointsZone diameters, 15 µg disc (mm) mg/L (≤S/>R) ≥SI≤R Enterobacteriaceae1/ Staphylococcus0.5/ Enterococcus0.25/ Streptococcus0.25/ Non-species related0.25/0.5---

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 H2OH2O 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 BreakpointsZone diameters (mm) mg/L (≤S/>R) ≥SI≤R Staphylococcus1/1--- [Enterococcus4/4]--- Streptococcus1/1--- [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.