Neil Woodford

Antibiotic Resistance Monitoring & Reference Laboratory, Centre for Infections

Antibiotic susceptibility testing: then, now and hereafter

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

CR-AB clones emerge

Resistance mechanisms

VRE

1985 1990 1995 20052000 20152010

Carbapenemases –

Enterobacteria;

NDM-1 discovered

CTX-M ESBL ‘explosion’

starts

Lin-R enterococci

VRE in animals

Dap-R staphs & enterococci

1st CTX-M ESBL

EMRSA

Woodford graduated

PCRGenome

sequence

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

1940s1940s 1946 Garrett:1946 Garrett:

multiple replication devicemultiple replication device concept of critical dilutionsconcept of critical dilutions

forerunner of agar-incorporation forerunner of agar-incorporation breakpointsbreakpoints

Modified by Steers et al 1959

1950s1950s

Comparative methodComparative method Joan Stokes 1955Joan Stokes 1955 Stokes & Waterworth 1972Stokes & Waterworth 1972 Stokes & Ridgway 1980Stokes & Ridgway 1980

Humphrey & Lightbown 1952

r2  =  9.21 Dt (logM – log 4πhDtc) r radius of the inhibitory zone t time from start c MIC D diffusion constant M disc potency H 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 CommitteesEuropean Committees

BSACUnited Kingdom

CA-SFMFrance

CLSI (NCCLS) USA

DINGermany

SRGASweden

CRGNetherlands

NWGANorway

StandardisationStandardisation 1959 Ericsson & Steers – evaluation of 1959 Ericsson & Steers – evaluation of

methodsmethods 1961 WHO – standardisation1961 WHO – standardisation 1964 Isenberg – comparison of methods in 1964 Isenberg – comparison of methods in

USAUSA 1964 Truant – standardised tube dilution MICs1964 Truant – standardised tube dilution MICs 1966 Bauer-Kirby1966 Bauer-Kirby

1975 NCCLS 1975 NCCLS CLSI CLSI 1998 BSAC Standardised Method 1998 BSAC Standardised Method 2009 EUCAST Standardised Method2009 EUCAST Standardised Method

EUCAST clinical MIC breakpoints

• Dosing, formulations

• Wild-type MIC distribution

• Existing national breakpoints

• PK/ PD data & Monte Carlo simulations

• Clinical data - outcome studies

• Tentative breakpoints are set for target species so as to avoid splitting the WT MIC distribution

• Consultation on proposed values

• Approval / publication on EUCAST website

”Dashed” – laboratories are recommended not to test

against this species

Click on name to directly access

MIC distributions

Insufficient evidence

Breakpoint tablesBreakpoint tablesavailable at http://www.eucast.orgavailable at http://www.eucast.org

”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 ?

MIC

Mechanism

Supplemental tests for Supplemental tests for mechanismsmechanisms

Hetero-resistance

• small sub-population of cells

• not easily detected

• some MRSA• h-VISA• colistin resistance

• may be distinct from full resistance

GISA

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

Black box approach:

molecular biology steps hidden

Simple end-product detection

Simple samplepreparation

• 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, web-based tools ?

• in the clinical diagnostic laboratory

– ↑ automated systems

– simple molecular methodologies

– tailored systems

– competitive market niche

Acknowledgements

Charles Easmon

Cathy Ison

Trevor Winstanley

Derek Brown

Robert George

David Livermore

ARMRL staff, 1988-present

Collaborators, 1985-present