Susceptibility testing new agents Dr Ian Morrissey Chief

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 vancomycinresistant 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) ≥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 Gramnegative 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) Zone diameters, 15 µg disc (mm) ≥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 H 2 O H 2 O & Oxyrase Aged MHB

Medium effect • Important for agar dilution or broth dilution. – Media >12 h 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) Zone diameters (mm) ≥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 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 van. A 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.