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 2

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

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

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

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

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 (www.bsac.org.uk) 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

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 10602 Ps. aeruginosa, NCTC 10418 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

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.

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

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

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

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

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

”Wild type” EUCAST determines epidemiological cut-off values for early detection of resistance ECOFF: WT ≤ 0.032 mg/L 15

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

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

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 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

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 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.

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

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

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