1. Antibiotic susceptibility testing: then, now and hereafter
Neil Woodford
Antibiotic Resistance Monitoring & Reference Laboratory, Centre for Infections

2. 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 2

3. Resistance mechanisms
Woodford graduated
CR-AB clones emerge
Carbapenemases –Enterobacteria;
NDM-1 discovered
VRE
1st CTX-M
ESBL
CTX-M ESBL ‘explosion’ starts
VRE in animals
Dap-R staphs & enterococci
Lin-R enterococci
EMRSA
Genome sequence
PCR
1985
1990
1995
2000
2005
2010
2015

4. AST How? Quantitative methods (MIC, mg/L) Qualitative methods (S I R)
Agar dilution
Broth dilution
Gradient methods
Qualitative methods (S I R)
Disk diffusion
Agar-incorporation breakpoint methods
Automated methods

5. The MIC is the lowest concentration of the antimicrobial required to inhibit growth of the organism.
Quantitative activity
The “gold standard” (not gradient methods)
Required for certain serious infections
endocarditis, pneumocococcal meningitis
Used for slow-growing organisms
􀁻It is used to determine the quantitative activity of an antimicrobial.
􀁻It is used to confirm resistance or equivocal results.
􀁻It is used in cases of prolonged treatment or endocarditis to adjust the dose of therapy.
􀁻It is used to determine the susceptibility of slow-growing organisms e.g anaerobes. 5

6. 1940s 1946 Garrett: multiple replication device
concept of critical dilutions
forerunner of agar-incorporation breakpoints
Modified by Steers et al 1959

7. Useful for laboratories for confirming resistance e. g
Useful for laboratories for confirming resistance e.g. penicillin resistance in S. pneumoniae
Are expensive therefore not used for first line testing
Caution:
Must use the MIC reading chart in the manual
BSAC methodology available on the BSAC web site (
A commercial alternative to tube MIC.
􀁻Consists of a plastic strip 6cm by 0.5cm in size.
􀁻Exponential gradient of antimicrobial dried on one side.
􀁻MIC scale printed on the other side.
Follow manufacturers’ instructions for inoculum preparation, media recommendations & incubation conditions.
􀁻MIC interpretation made where growth of inhibition ellipses the strip.
􀁻Most E-test require examination with a hand-lens to look for minute colonies intersecting the strip.
􀁻The range corresponds to fifteen 2-fold dilutions.
In the lab we use E-tests routinely to check:-
􀁻Mupiriocin susceptibility (MRSA).
􀁻Confirmation of teicoplanin and/or vancomycin resistance (GISA, VRE).
􀁻Isolates of Salmonella typhi / paratyphiciprofloxacin susceptibility.
􀁻Confirmation of penicillin resistant Streptococcus pneumoniae.
􀁻Helicobacter pylori susceptibility.
􀁻Yeast fluconazole susceptibility. 7

8. 1950s Comparative method Joan Stokes 1955 Stokes & Waterworth 1972
Stokes & Ridgway 1980
Introduced in the late 1960s
Novel method that allowed control of test performance at a time when laboratories made their own media and discs
Advantage???
Laboratories tested any disc the microbiologist asked for using the same criteria for interpreting susceptibility
Disadvantages
No correlation with clinical response
Not upgraded as new antibiotics introduced
iii Every laboratory had its own version of method
iv Unsuitable for surveillance of resistance studies
v New more potent antibiotics often interpreted as falsely resistant
Developed in the U.K (1972).
􀁻A variety of media can be used including Iso-sensitest agar (ISA), ISA & 5% lysed blood & Chocolate ISA.
􀁻Based on dense not confluent growth.
􀁻Use suspension of organism in broth equivalent in density to an overnight broth culture.
􀁻Inoculate fastidious orgs direct
Use NCTC (National Collection of Type Cultures) controls e.g. NCTC 6571 Staph aureus, NCTC Ps. aeruginosa, NCTC E.coli.
􀁻Using a rotary plater apply the control suspension on the outer edge & the test suspension in the centre, leaving a gap for the discs
Interpretation based on comparison between zones seen with the test organism & those of the known sensitive control.
􀁻Sensitive = zone radius of test, equal, or not more than 3mm smaller than the control.
􀁻Intermediate = zone radius more than 3mm, but smaller than the control by more than 3mm.
􀁻Resistant = zone radius of 3mm or less.
Interpretation not valid for penicillinase-producing staphylococci (research for practical next week) or for tests with polymyxin, augmentin, teicoplanin or ciprofloxacin.
􀁻No correlation of zone diameter with the MIC of the organism.
􀁻No standard method for inoculum preparation-a heavy inoculum decreases zone of inhibition.
No standard method for media or incubation conditions-pre-incubation decreases the zone of inhibition, pre-diffusion increases the zone of inhibition.
􀁻Unreliable for detection of resistance to new antibiotics or newer resistance mechanisms (ESBL’s).
􀁻No consistent method between labs, therefore no consistent epidemiology data. 8

9. 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
By altering disc potency, the zone diameter can be altered to a suitable size.

10. 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) 10

11. European Committees BSAC SRGA CA-SFM CRG DIN NWGA United Kingdom
Sweden
CA-SFM
France
CRG
Netherlands
DIN
Germany
NWGA
Norway
CLSI (NCCLS) USA

12. Standardisation 1959 Ericsson & Steers – evaluation of methods
1961 WHO – standardisation
1964 Isenberg – comparison of methods in USA
1964 Truant – standardised tube dilution MICs
1966 Bauer-Kirby
1975 NCCLS  CLSI
1998 BSAC Standardised Method
2009 EUCAST Standardised Method
WRG Netherlands

13. 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

14. 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 14

15. ”Wild type”
EUCAST determines epidemiological cut-off values for early detection of resistance
ECOFF: WT ≤ mg/L 15

16. EUCAST compared with CLSI
Committee representing the medical profession and Science with input from Regulatory (ECDC, EMEA).
Financed by ESCMID and ECDC
Industry consultative role
Consensus process with the profession as drivers
Five meetings per year.
EUCAST is the breakpoint committee of EMEA
Transparent, rationale documents provided
Documents for free!
Clinical breakpoints and epidemiological cut-offs
Committee representing Industry, the medical profession, science and Regulatory
Financed by incomes from industry and documents
Industry major influence on decision process
Voting by committee members
Two meetings per year.
CLSI is not consulted by FDA and there is a 24 month ban.
No published rationale for decisions
Documents for sale!
Clinical breakpoints 16

17. Adding value to AST… Interpretative reading it’s not an exact science
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

18. Interpretative reading
Examine the whole phenotype
Apply “expert” knowledge
…, but you must
Identify to species
Test a large panel of
antibiotics

19. All in a box: automated AST +/- ‘expert’ interpretation

20. What’s more important for appropriate therapy ?
Mechanism
MIC

21. Supplemental tests for mechanisms

22. Hetero-resistance GISA Hetero-GISA GSSA small sub-population of cells
not easily detected
some MRSA
h-VISA
colistin resistance
may be distinct from full resistance
GISA
Hetero-GISA
GSSA

23. 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

24. 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

25. Molecular detection Simple and multiplex PCR Real-time PCR
DNA sequencing
Hybridisation-based techniques
Identibac AMR- is a quick and simple method for detecting and typing antimicrobial resistance in gram negative bacteria. The Identibac AMR- ArrayTube contains probes for 54 different anti microbial resistance genes from gram negative bacteria. Idenitbac AMR- allows identification of genes in clinical isolates of gram negative bacteria of human and animal origin that correspond to a range of antimicrobial resistance phenotypes including: quinolones, sulphonamides, tetracyclines, integrases, aminoglycosides, carbenicillinase, chloramphenicols, trimethoprims, plasmidic Amp C, and beta-lactams.
Identibac SA is a quick and simple method for identifying antimicrobial resistance and virulence factors in Staphylococcus aureus strains. The Identibac SA ArrayTube contains 39 different probes for detecting 29 different genes or gene groups associated with antimicrobial resistance and virulence factors in S. aureus. Identibac SA allows identification of virulence genes associated with pathotypes including: toxic shock syndrome, panton valentine leukocidin, entertoxins, and staph. scalded skin syndrome. Identibac SA also detects genes associated with resistance to a range of different antimicrobials including: aminoglycoside, trimethoprim, tetracycline, streptogramin, beta-lactamase, methicillin, penicillin, streptothricin, vancomycin, teicoplanin, mupriocin, and fusicidic acid.

26. 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

27. more cost-effective than PCR
Chips with everything…
going beyond AST !
Total profiling;
more cost-effective than PCR
species identification
resistance genes
virulence genes
epidemicity predictors
strain-specific markers

28. 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

29. 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

30. Acknowledgements Charles Easmon Cathy Ison Trevor Winstanley
Derek Brown
Robert George
David Livermore
ARMRL staff, 1988-present
Collaborators, 1985-present