Standardisation – What does this mean for the CDS?

Keys to Standardisation Agreement on MIC breakpoints that separate susceptible and resistant strains Each primary method should be calibrated to a gold standard minimum inhibitory concentration method (GSMIC). Concordance of results using international reference strains. Support for the diagnostic laboratory.

What is the goal of performing antimicrobial susceptibility testing? To predict the in vivo success or failure of antibiotic treatment. In vitro testing performed. Standardised CDS primary testing ensures reproducibility.

What is susceptible? Susceptible category indicates that the organism is likely to have a favourable response to treatment with the tested antibiotic at the recommended dosage.

Comparison of MIC Breakpoints S aureus (21), Enterobacteriaceae (29), Ps aeruginosa (13) n=63 Breakpoints (S) CDS EUCAST CLSI Concordant 60 48 57 Discordant 3 9(6) 2(4)

Gold standard MIC CDS calibrated using agar dilution WHO accepts agar dilution as a gold standard reference method for calibration of antibiotic disc testing (CDS & CLSI) Broth microdilution MIC ISO 20776-1 introduced in 2007. EUCAST calibrated using Broth microdilution and gradient diffusion.

Harmonisation Various GCMIC methods must yield similar results. Demonstrated by concordance using international reference strains. NPACC requested harmonisation project.

Comparison of QC strains MIC Antibiotics CDS Agar CLSI Agar EUCAST Broth Concordant 57 56 Discordant 2 3 E faecalis ATCC 29212 (11), S aureus ATCC 29213 (21), E coli ATCC 25922 (14) Ps aeruginosa ATCC 27853 (13) n=59

Support for the diagnostic laboratory Confidence in results Competence in external audit CDS Reference Laboratory

Back to Basics with the CDS Antibiotic Susceptibility Test ASM Hobart 2017 Julie Allerton

Overview History of development of CDS Features Support of the Method Calibration Uniform cut-off Dichotomous reporting as S/R Compliance with the method Support of the Method The manual The Reference Laboratory The website

History 1968-1973 – RCPA demonstrates considerable inaccuracy in reporting of antibiotic susceptibility 1971 – up to 27% error in reporting penicillin resistant staphylococci 1973 – staphylococci reported without error using CDS method 1975 – CDS Test published in “Pathology” to fulfil need for a simple calibrated method

Calibration Provides a confident scientific prediction of likely response Plotting observed zone size against the log of MIC of antibiotic Agar dilution method of Ericsson and Sherris (WHO gold standard)

Annular Radius

Uniform Cut-off Interpretation of results are simplified and less prone to error Similar zone of inhibition achievable with most antibiotics >6mm – susceptible <6mm – resistant Positive predictive value >98% - “true susceptible”

Diffusion Sigmoid Curve Point on curve enabling greatest discrimination between S/R

Dichotomous Reporting Susceptible or resistant Bi-modal distribution Separation point of 6mm annular radius in disc testing

Bi-modal Distribution

Continuous Distribution

Compliance with Method Strict adherence to method QANTAS checklist Weekly quality assurance 95% confidence limit

The Manual Unique features of the test and quality assurance Regulatory requirements Tables for calibrations, surrogate disc testing, reporting of β-lactams for Gram negatives Application to unusual organisms

“This organism has not been calibrated by the CDS method “This organism has not been calibrated by the CDS method. The results have been extrapolated and are provisional only”.

The Reference Laboratory Provides local support to users and expert advice Able to calibrate novel agents Reactive to changes Regular updates Supply reference strains with quality certificates Free of charge

The Website Most current version Notifications via registered users list www.cdstest.net Contact us for assistance with any CDS matter

Contact Details The CDS Reference Laboratory Department of Microbiology (SEALS) Clinical Services Building St. George Hospital Kogarah NSW 2217 Australia Tel: (02) 9113 3346 Fax: (02) 9113 3349 Dianne.Rafferty@health.nsw.gov.au Julie.Allerton@health.nsw.gov.au NSWPATH-SealsCDS@health.nsw.gov.au

Thank you I would like to acknowledge the following people for their on-going help and support Prof. Sydney Bell A/Prof Peter Taylor Dianne Rafferty

CDS Update and review of Antimicrobial Resistance ASM Hobart 2017 Antimicrobial Special Interest Group  Dianne Rafferty

Overview of CDS CDS uses 107 cfu/mL inoculation Flood plates Uses low disc concentrations This combination allows observation of not only the zone size but also its morphology Variation of Phenotypic expression of enzymes.

Haemophilus influenzae Mechanisms of Resistance: Production of β-lactamase (TEM type or ROB-1) Altered PBPs (BLNAR) Combination of the two (BLPACR)

Testing and Reporting H. influenzae BLNAR & BLPACR β-lactamase positive: R/Ampicillin (Amp5) only and can be confirmed by Nitrocefin hydrolysis. BLNAR:R/Ampicillin (Amp5) Cefotaxime (CTX 0.5) or ceftriaxone (CRO 0.5) < 6mm Cefotaxime (CTX 5) or ceftriaxone 0.5ug (CRO 0.5) ≥ 6mm BLPACR: R/ampicillin (Amp5), Augmentin (AMC15)

β-lactamase positive H influenzae No zone of inhibition Ampicillin Both potencies of CTX >6mm β-lactamase positive H influenzae

Testing and Reporting H. influenzae BLNAR & BLPACR β-lactamase positive: R/Ampicillin (Amp5) only and can be confirmed by Nitrocefin hydrolysis. BLNAR:R/Ampicillin (Amp5) Cefotaxime (CTX 0.5) or ceftriaxone (CRO 0.5) < 6mm Cefotaxime (CTX 5) or ceftriaxone 0.5ug (CRO 0.5) ≥ 6mm BLPACR: R/Ampicillin (Amp5), Augmentin (AMC15)

R/Ampicillin CTX 5ug ≥6mm CTX 0.5ug <6mm H influenzae with BLNAR Report as ‘There is decreased susceptibility to cefotaxime with the MIC between 0.5mg/L and 2.0 mg/L’.

Testing and Reporting H. influenzae BLNAR & BLPACR β-lactamase positive: R/Ampicillin (Amp5) only and can be confirmed by Nitrocefin hydrolysis. BLNAR:R/Ampicillin (Amp5) Cefotaxime (CTX 0.5) or ceftriaxone (CRO 0.5) < 6mm Cefotaxime (CTX 5) or ceftriaxone 0.5ug (CRO 0.5) ≥ 6mm BLPACR: R/Ampicillin (Amp5), Augmentin (AMC15)

R/Ampicillin CTX 5ug ≥6mm CTX 0.5ug <6mm H influenzae with BLPACR Report as ‘There is decreased susceptibility to cefotaxime with the MIC between 0.5mg/L and 2.0 mg/L’.

Classification of β-lactamases Ambler class Bush group (molecular structure) (inhibitor) A (eg. TEM-1, SHV-1) Group 2 (inhibited by CA) B (MBLs eg. IMP,VIM, NDM) Group 3 – Metallo Zn (not inhibited by CA inhibited by EDTA) C (AmpC eg. CMY,ACT,FOX,DHA) Group 1 (not inhibited by CA, inhibited by boronic acid) D (OXA eg. OXA-48, OXA-23) Group 4 (A. baumanii)

(Ambler class A, Bush group 2) (no need for confirmation) ESBLs (Ambler class A, Bush group 2) Inhibited by CA R/ Cephalosporins (including cefepime) and aztreonam S/ Augmentin (AMC 60) S/ Cephamycin (cefoxitin, cefotetan) CDS routine testing → Synergy with AMC 60 (no need for confirmation) S/ Imipenem (T)

K. pneumoniae producing an ESBL S/ Augmentin (AMC 60), typical synergy with cephalexin (CL 100) and cefotaxime (CTX 5). S/ imipenem (IMP 10) and cefotetan (CTT 30).

CDS Routine Testing: Positioning strongly recommended (urine isolate) CDS Routine Testing: Positioning strongly recommended (urine isolate). Klebsiella pneumoniae producing an ESBL: synergy between Augmentin (ACM 60) and cefepime (FEP 10). High activity => no obvious synergy with cefotaxime (CTX 5).

Acquired Metallo-Beta-Lactamases (MBLs) Ambler class B or Bush group 3 Plasmid mediated MBLs Ambler class B or Bush group 3 Inhibited by EDTA (Zinc molecule) IMP-4 (most common) VIM, SPM, GIM, SIM (P. aeruginosa) Hydrolyses all beta-lactam (except aztreonam) Enterobacteriaceae May have a zone > 6mm with IPM 10 Pseudomonas aeruginosa Highly resistant to all β-lactams (S/ATM)

C. diversus R/AMC 60, CL100, CTX 5 and colonies in cefepime zone (FEP 10) and some at the edge of imipenem zone (> 6 mm). No synergy between FEP/AMC → not ESBL Numerous resistant colonies in FEP 10 → Candidate for MBL detection

Simple detection of MBL: Same isolate showing synergy between an EDTA disc (blank) placed next to cefotaxime (CTX 5)/ imipenem (IPM 10)/ cefepime (FEP 10)/ Augmentin (AMC 60) discs.

=> MBL (IMP or NDM) and ESBL Klebsiella pneumoniae: Synergy between EDTA (blank discs) and imipenem (IPM 10), ertapenem (ETP 10) R/ aztreonam (ATM30) and synergy with Augmentin (AMC 60) => MBL (IMP or NDM) and ESBL

Plasmid mediated and chromosomal Types Of AmpC Plasmid mediated and chromosomal E.coli, K.pneumo EEC group, S.marcescens, Salmonella, H.alvei, P.stuartii, P.rettgeri, M.morganii

PM-AmpC in E. coli and Klebsiella R/ AMC 60 (not inhibited by CA) R/ CL 100 S/ FEP 10 Standard interpretation > 6 mm (no resistant col.) => S/ CTX 5 < 6 mm => R/ CTX 5 Confirmation (optional): inhibition by boronic acid (BA) (1-Benzothiophene-2-boronic acid)

cephalexin (CL), cefotaxime (CTX) Routine CDS test showing an E. coli with a TEM-1 and a plasmid mediated AmpC. R/ ampicillin (AMP) Augmentin (AMC), cephalexin (CL), cefotaxime (CTX) S/ cefepime (FEP 10) and imipenem (IPM 10).

Confirmation of AmpC with Boronic Acid Same E.coli showing synergy between boronic acid discs (blank) and adjacent cefotaxime (CTX 5) , Augmentin (AMC 60), cephalexin (CL 100) and ceftazidime (CAZ 30)

Chromosomal Inducible AmpC Four distinct sub groups Flattened inhibitory zone High mutation rate R/CL, CTX, AMC R/ATM

Oxa-48 and Oxa-181 No definitive Phenotypic test only Presumptive ID Imipenem annular radius (AR)<6mm or 6-7 mm with mutant colonies at zone edge Plus Resistant to Augmentin & Tazocin Cefotaxime/Ceftriaxone & Cefepime AR > carbapenems Perform further confirmatory tests Whilst these highly resistant strains cannot be detected by phenotypic techniques, they will be recognized as resistant to all carbapenems in routine CDS testing, providing the methodological process is closely followed.

E. coli Oxa-48 – Confirmation required

Summary of Beta lactamases Ambler Group Inhibitor AMC60 FEP10 CTT30 ESBL A Clavulanic acid S R MBL B EDTA NT PM AmpC C Boronic Acid OXA D None

Pseudomonas aeruginosa producing an ESBL in routine CDS test S/ Timentin (TIM 85), typical synergy between Timentin and ceftazidime (CAZ 10)

No synergy Pseudomonas aeruginosa candidate for MBL detection: highly resistant to all β-lactams, imipenem (IPM 10), meropenem (MEM 5), ceftazidime (CAZ 10), tazocin (TZP 55), cefepime (FEP 10) and Timentin (TIM 85).

Detection of MBL: Synergy between an EDTA disc (blank disc) placed next to imipenem (IMP 10), tazocin (TZP 55), Timentin (TIM 85), ceftazidime (CAZ10), S/aztreonam (ATM30)

Pseudomonas aeruginosa: highly resistant to all β-lactams, imipenem (IPM 10), meropenem (MEM 5), ceftazidime (CAZ 10), tazocin (TZP 55), cefepime (FEP 10) and Timentin (TIM 85). ?MBL

Detection of MBL: Non-specific synergy – NOT MBL

CarbaNP negative result suggesting GES present confirm with molecular testing.

Comparison of Boronic acid results: Non-specific (left) and MBL +ve strain (right).

armA – Aminoglycoside resistance methylase gene High level resistance all aminoglycosides except streptomycin Plasmid mediated First found in Klebsiella pneumoniae Gaimand et at (2005) found highly associated with CTX-M (ESBL) CARAlert

Klebsiella pneumoniae ESBL demonstrated ?armA present ESBL Detected R/aminoglycosides R/aminoglycoside Klebsiella pneumoniae ESBL demonstrated ?armA present

Klebsiella pneumoniae resistant to all aminoglycosides Klebsiella pneumoniae resistant to all aminoglycosides. Molecular confirmation required.

Klebsiella pneumoniae – R/AMC 60, no obvious keyhole, R/cephalosporins, R/FEP, boarder line zone IPM with colonies at the edge. Could this be ESBL/MBL or both?

Klebsiella pneumoniae showing ESBL with synergy between Augmentin (AMC60)/aztreonam (ATM30), MBL demonstrated by the synergy between EDTA (blank discs) and imipenem (IMP10) and ertapenem (ETP 10)

E. coli isolate R/Augmentin (AMC 60), no synergy demonstrated, R/cephalexin (CL 100), Cefotaxime (CTX 5) with colonies, S/Cefepime (FEP 10) and S/imipenem (IPM 10)

E coli isolate – no ESBL demonstrated E coli isolate – no ESBL demonstrated. Colonies present in cefotaxime zone ?ampC – set up boronic acid

E coli: Confirmation of ampC with boronic acid (blank discs) showing synergy with cefotaxime CTX, cephalexin (CL), and ceftazidime (CAZ)

Enterobacter kobei (part of the Enterobacter cloacae complex, EEC subgroup): R/cefotetan (CTT30), R/aztreonam (ATM 30). Both cefepime and imipenem have large zones with colonies at the edge. ?ampC and ESBL present

Enterobacteria kobei investigation for ESBL

Enterobacter kobei Boronic acid test showing the presence of ampC

Conclusion CDS proven and practical susceptibility test CDS can readily demonstrate phenotypic expression of resistant mechanisms Confirmation can be performed using alternative methods. Presence of a gene does not always equate to phenotypic expression Other factors such as porin loss and efflux pumps can affect susceptibility patterns

Acknowledgements Special thanks to Professor Bell and Julie Allerton for their guidance and support SEALS Kogarah, especially A/Prof Peter Taylor and Chinmoy Mukerjee