1. Standardisation – What does this mean for the CDS?

2. 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.

3. 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.

4. 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.

5. 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)

6. 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 introduced in 2007.
EUCAST calibrated using Broth microdilution and gradient diffusion.

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

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

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

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

11. 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

12. History
– 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

13. 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)

14. Annular Radius

15. 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”

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

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

18. Bi-modal Distribution

19. Continuous Distribution

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

21. 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

22. “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”.

23. 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

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

25. Contact Details
The CDS Reference Laboratory Department of Microbiology (SEALS) Clinical Services Building St. George Hospital Kogarah NSW Australia Tel: (02) Fax: (02)

26. 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

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

28. 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.

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

30. 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)

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

32. 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)

33. 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’.

34. 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)

35. 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’.

36. 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)

37. (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)

38. 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).

39. 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).

40. 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)

41. 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

42. 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.

43. => 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

44. 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

45. 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)

46. 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).

47. 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)

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

49. 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.

50. E. coli Oxa-48 – Confirmation required

51. 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

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

53. 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).

54. 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)

55. 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

56. Detection of MBL: Non-specific synergy – NOT MBL

57. CarbaNP negative result suggesting GES present confirm with molecular testing.

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

59. 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

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

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

62. 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?

63. 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)

64. 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)

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

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

67. 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

68. Enterobacteria kobei investigation for ESBL

69. Enterobacter kobei Boronic acid test showing the presence of ampC

70. 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

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