Antibiotic susceptibility testing:
then, now and hereafter
Neil Woodford
Antibiotic Resistance Monitoring & Reference Laboratory,
Centre for Infections
AST Why?
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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?
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Quantitative methods (MIC, mg/L)
Agar dilution
 Broth dilution
 Gradient methods
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Qualitative methods (S I R)
Disk diffusion
 Agar-incorporation breakpoint methods
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Automated methods
1940s
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1946 Garrett:
multiple replication device
 concept of critical dilutions
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forerunner of agar-incorporation breakpoints
Modified by Steers et al 1959
1950s
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Comparative method
Joan Stokes 1955
 Stokes & Waterworth 1972
 Stokes & Ridgway 1980
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Humphrey & Lightbown 1952
 r2
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= 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
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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
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1959
1961
1964
1964
1966
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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
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Examine the whole phenotype
Apply “expert” knowledge
…, but you must
Identify to species
 Test a large panel of
antibiotics
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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
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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