Antibiotic susceptibility testing: then, now and hereafter Neil Woodford Antibiotic Resistance Monitoring & Reference Laboratory, Centre for Infections AST Why?         Cannot assume susceptibility or resistance Guidance for treatment of the individual patient Background information for empirical treatment To set local and national prescribing policies To monitor epidemiological trends For surveillance of resistance To test the activity of new agents Means of detecting new resistances Resistance mechanisms Woodford graduated CR-AB clones emerge VRE 1st CTX-M ESBL VRE in animals Lin-R enterococci EMRSA Dap-R staphs & enterococci Genome sequence PCR 1985 CTX-M ESBL ‘explosion’ starts Carbapenemases –Enterobacteria; NDM-1 discovered 1990 1995 2000 2005 2010 2015 AST How?  Quantitative methods (MIC, mg/L) Agar dilution  Broth dilution  Gradient methods   Qualitative methods (S I R) Disk diffusion  Agar-incorporation breakpoint methods   Automated methods 1940s  1946 Garrett: multiple replication device  concept of critical dilutions   forerunner of agar-incorporation breakpoints Modified by Steers et al 1959 1950s  Comparative method Joan Stokes 1955  Stokes & Waterworth 1972  Stokes & Ridgway 1980  Humphrey & Lightbown 1952  r2        = 9.21 Dt (logM – log 4πhDtc) r t c D M H radius of the inhibitory zone time from start MIC diffusion constant disc potency depth of agar Zone size is: directly proportional to the diffusion constant  directly proportional to the log of the disc potency  inversely proportional to the log of the MIC  MICs and zone sizes are meaningless …unless you apply interpretative criteria clinical breakpoints indicate likelihood of therapeutic success (S) or failure (R ) of antibiotic treatment based on microbiological findings (S≤ Y mg/L and R> Z mg/L) epidemiological cut-off values (ECOFFs) separate microorganisms without (wild type) and with acquired or mutational resistance (non-wild type) (WT≤ X mg/L) European Committees BSAC SRGA United Kingdom Sweden CA-SFM CRG France Netherlands DIN NWGA Germany Norway CLSI (NCCLS) USA Standardisation      1959 1961 1964 1964 1966    Ericsson & Steers – evaluation of methods WHO – standardisation Isenberg – comparison of methods in USA Truant – standardised tube dilution MICs Bauer-Kirby 1975 NCCLS  CLSI 1998 BSAC Standardised Method 2009 EUCAST Standardised Method EUCAST clinical MIC breakpoints • Existing national breakpoints • Tentative breakpoints are set for target species so as to avoid splitting the WT MIC distribution • PK/ PD data & Monte Carlo simulations • Consultation on proposed values • Clinical data - outcome studies • Approval / publication on EUCAST website • Dosing, formulations • Wild-type MIC distribution Breakpoint tables available at http://www.eucast.org Click on name to directly access MIC distributions ”Dashed” – laboratories are recommended not to test against this species Insufficient evidence ”Wild type” EUCAST determines epidemiological cut-off values for early detection of resistance ECOFF: WT ≤ 0.032 mg/L Adding value to AST… • Interpretative reading – Infer mechanisms from patterns (antibiograms) – Recognise grossly unusual – Edit susceptibilities / identify further drugs to test – Tentative surveillance of resistance mechanisms • it’s not an exact science – there are always exceptions and anomalies Interpretative reading    Examine the whole phenotype Apply “expert” knowledge …, but you must Identify to species  Test a large panel of antibiotics  All in a box: automated AST +/‘expert’ interpretation What’s more important for appropriate therapy ? Mechanism MIC Supplemental tests for mechanisms Hetero-resistance • small sub-population of cells GISA • not easily detected • some MRSA • h-VISA • colistin resistance • may be distinct from full resistance Hetero-GISA GSSA Molecular detection: where and why ? • In the Reference Laboratory – confirmation of unusual resistance – surveillance of resistance mechanisms – monitoring spread of resistance genes / strains – identify strains likely to contain novel resistance mechanisms • In the clinical diagnostic laboratory – rapid detection for patient management – infection control Molecular detection: different needs • In the Reference Laboratory, – testing “pure” cultures – myriad assays and formats – numerous bug-drug combinations • In the clinical diagnostic laboratory – directly from specimens – need to target key species – format must be simple, rapid and cost-effective – problems with genes in commensals Molecular detection     Simple and multiplex PCR Real-time PCR DNA sequencing Hybridisation-based techniques Molecular tests in clinical labs Simple sample preparation Black box approach: molecular biology steps hidden Simple end-product detection • Must be rapid (TATs), inexpensive, reliable ! • Platform must be sufficiently versatile to justify investment • Relatively hands-free, with scope for automation • On-going – e.g. <30 min test for ESBL detection Chips with everything… going beyond AST ! Total profiling; more cost-effective than PCR • species identification • resistance genes • virulence genes • epidemicity predictors • strain-specific markers Molecular detection: the inherent problem • Molecular methods only detect known mechanisms • only as good as available sequence data • resistant isolates with known genes identified • & new variants, if sufficient homology • false-resistance (unexpressed / partial genes) • Susceptibility must always be confirmed • can’t base treatment on a negative molecular result • can’t detect genuinely new resistance mechanisms • will never (?) replace cheap phenotypic methods What next for AST ? • in the Reference Laboratory – increased use of arrays, especially to support surveys, neural networks, webbased tools ? • in the clinical diagnostic laboratory – ↑ automated systems – simple molecular methodologies – tailored systems – competitive market niche Acknowledgements Charles Easmon Robert George Cathy Ison David Livermore Trevor Winstanley ARMRL staff, 1988-present Derek Brown Collaborators, 1985-present