Mini-Review presentation Best Practice for Colistin Susceptibility Testing: Methods and Evidence Clinical Microbiology Residency Program, King Fahd Hospital of the University Al Khobar, Saudi Arabia

1. Best Practice for Colistin
Susceptibility Testing: Methods and
Evidence
Abdullatif Sami Al Rashed
Microbiology Resident
Teaching Assistant, Department of Microbiology,
College of Medicine, Imam Abdulrahman Bin
Faisal University
Dammam, Saudi Arabia

2. Outline
• Introduction
– History.
– Mechanism of Action.
– Spectrum of Activity.
– Mechanism of Resistance.
• Colistin AST
– Background

3. INTRODUCTION

4. Introduction
Colistin is a cationic polypeptide
antibiotic that is a member of the
polymyxin class of antibiotic.
This drug was first identified from the
isolation of bacterium Paenibacillus
polymyxa subsp. colistinus.

5. History
This restriction was essential because of its toxicity, especially
nephrotoxicity.
However, during the 1970s, the usage of colistin was restricted and
minimized only for ophthalmic and topical use.
For many decades, colistin was extensively used to treat Gram-
negative bacterial infections.
Discovered initially in 1947, and used clinically in Europe and Japan
during the 1950s.
Colistin is considered as one of the first antibiotics introduced as a
1ST line treatment of severe Gram-negative bacterial infections.

6. History
However, the growing
evolution of multidrug-
resistant (MDR) Gram-
negative bacterial
infections has forced
physicians to once again
use this antibiotic as a
primary treatment option.

8. Mechanism of Action
• The primary target site of this antibiotic is the
lipopolysaccharide (LPS) part of the outer
membrane.
• Colistin attaches to the negatively charged cell
wall and leads to the destruction of the
bacterial outer membrane, thus leading to
bacterial cell death. (bactericidal antibiotic)

9. Spectrum of Activity
• In general, colistin has a narrow antimicrobial
spectrum which are toward Gram-negative
bacteria primarily against:
Enterobacteriaceae
S.
maltophilia
A.
baumannii
P.
aeruginosa

10. Mechanism of Resistance
• Both chromosomal and plasmid-encoded
resistance genes have been identified in many
genera of Enterobacteriaceae against colistin.
• However, many resistance mechanisms for
other bacterial species remain undiscovered.

11. Mechanism of Resistance
• Modification of the outer membrane
lipopolysaccharide target of is the well-known
resistance mechanism of colistin.
• mcr-1 and mcr-2 genes are relatively newly
discovered (discovered in 2016) plasmid-borne
mobilized genes found in Enterobacteriaceae that
are inducing horizontal transmission of colistin
resistance.
• Recently, Three additional plasmid borne
mobilized genes were identified mcr-3, mcr-4, mcr-
5

12. Venter H. et al, 2017

13. Manual of clinical microbiology 12th edition

14. MCR-1-producing isolates reported in humans, animals, and
both humans and animals. (Poirel L et al, 2017)

15. Intrinsic Resistance
• Several gram negative bacterial species are
intrinsic resistant to colistin including:
1. B. cepacia and B. mallei,
2. Brucella spp. & Neisseria spp.
3. Providencia spp. & M. morganii,
4. V. cholerae,
5. Chromobacterium spp.,
6. S. marcescens, Edwardsiella spp.,
7. Legionella spp., and Campylobacter spp.

16. AIM
• Understanding the mechanisms of resistance and
the genes responsible for these mechanisms is
very crucial for determining the most suitable
method of antimicrobial susceptibility testing to
colistin and subsequently decreasing treatment
failure rates.
• This mini review aims to discuss and address the
current and latest updates in colistin
antimicrobial susceptibility testing.

17. COLISTIN ANTIMICROBIAL
SUSCEPTIBILITY TESTING

18. Background of AST
• Colistin susceptibility testing is considered a
big challenge, as it faces several difficulties
including:
– Its poor and slow diffusion into agar,
– its natural cationic features,
– The exhibited hetero-resistance of multiple
species to this antibiotic, and
– The need of an easy and dependable reference
standard testing that could provide constant
comparisons of commercial testing systems.

19. Background of AST
• In July 2016, the European Committee on
Antimicrobial Susceptibility Testing (EUCAST)
published a warning letter regarding problems arising
in various commercially available colistin
susceptibility tests.

20. Background of AST
Summary of the warning:
• Disk diffusion cannot be used for susceptibility testing
of colistin. It does not discriminate between susceptible
and resistant isolates.
• Currently available gradient tests underestimate colistin
MIC values and undercall resistance, and should be
avoided, even when quality control results are within
range.
• The single valid and reliable method of colistin antimicrobial
susceptibility testing is broth microdilution.
http://www.eucast.org/fileadmin/src/media/PDFs/E
UCAST_files/Warnings/Warnings_docs/Warning_-
_colistin_AST.pdf

21. Background of AST
• The poor and slow diffusion of polymyxins
through agar in disc diffusion is associated
with small zones of growth inhibition and
significant assay variation
• The predictive accuracy of the DD test is
unacceptable, and consequently, no reliable
correlation of zone diameters and MICs has
been found in many previous studies

22. Reference Standard Method
Joint
EUCAST/CLSI
working group
recommends
the use of the
broth
microdilution
ISO-74 20776
for the MIC
measurement
of colistin.
These
recommendations
specifically
address using
cation-adjusted
Mueller Hinton
broth, and adding
of the colistin
sulfate salts in a
plain polystyrene
tray with no
additives, such as
polysorbate-80, or
any similar
surfactant.
Use of
methanesulfon
ate derivative
salts instead of
sulfate salts
should be
avoided, due to
the poor
dissolution of
colistin in it.
https://www.iso.org/standard/41630.html

23. Reference Standard Method
• A significant limitation in broth microdilution is
the microtiter plate attachment of colistin.
• To counter that, a surfactant such as polysorbate-
80 is added to reduce the bonding of colistin to
the microtiter plates.
• However, this has been shown to lead to a
synergistic effect with colistin due to the innate
antibacterial activity of Polysorbate-80.
• Therefore, neither EUCAST nor CLSI recommend
adding polysorbate-80 to the microtiter plates.

24. Breakpoints
• The 2019 published International Consensus
Guidelines for the Optimal Use of the
Polymyxins, recommends following the joint
EUCAST/CLSI working group colistin clinical
breakpoints for Acinetobacter baumanni and
Pseudomonas aeruginosa (Table 1).

25. Breakpoints
Because of absence of adequate
evidence in the literature regarding
colistin breakpoints in
Enterobacteriaceae, CLSI
recommends using the
epidemiological cut-off value (ECV)
rather than the clinical breakpoints
for E. coli, K. aerogenes, R.
ornithinolytica, E. cloacae, and K.
pneumoniae (Table 2).
On the contrary, EUCAST
has published specific
colistin breakpoints for
Enterobacteriaceae (Table
1)

26. Table 1. CLSI/EUCAST Breakpoints for Colistin

27. Table 2. CLSI Recommendation of Enterobacteriaceae ECV for Colistin

28. Despite BMD being the
only method that is
recommended to be
used for colistin
antimicrobial
susceptibility testing in
clinical microbiology
labs, yet, the fact that
this method is
considered difficult and
time-consuming to be
done on a daily basis,
limits its application.

29. Colistin AST
• Moreover, some reports have demonstrated that
certain bacterial species have non-interpretable and
non-reproducible MIC values due to skip wells
– Skip wells phenomenon is demonstrated when wells
shows no growth, while growth can be detected in wells
with greater antibiotic concentrations, suggesting hetero-
resistant strains.
– This phenomenon was specifically associated with P.
aeruginosa, A. baumannii, and Enterobacter species.
• Accordingly, most clinical microbiology laboratories do
not perform antimicrobial susceptibility testing for
colistin with the traditional BMD method.

30. COMMERCIAL BROTH MICRODILUTION
METHOD SYSTEMS

31. Background
Nowadays, multiple different commercial
BMD systems are available.
They are practical and uncomplicated in
comparison to the traditional BMD, and
recently, several studies have evaluated
them.

32. Matuschek et al, 2018
• Matuschek et al. have evaluated five
commercial BMD systems and 2 gradient tests
in comparison to the BMD reference standard
method (ISO standard 20776-1)
Matuschek et al, Clinical microbiology and infection.
2018

33. Matuschek et al, 2018
• The 5 commercial systems tested were:
1. MICRONAUT MIC-Strip (MERLIN Diagnostika
Gmbh, Bornheim, Germany),
https://www.merlin-
diagnostika.de/fileadmin/mediapool/downloads/Produkte/Resistenzbestimmung/1858845_
MIC-STRIP_Colistin_Flyer_04_2018_ebook.pdf

34. Matuschek et al, 2018
• The 5 commercial systems tested were:
2. MICRONAUT-S (MERLIN Diagnostika Gmbh,
Bornheim, Germany)
https://www.merlin-
diagnostika.de/fileadmin/mediapool/downloads/Produkte/Resistenzbestimmung/F_MCN-

35. Matuschek et al, 2018
• The 5 commercial systems tested were:
3. SensiTest (Liofilchem, Roseto degli Abruzzi, Italy)
https://www.liofilchem.com/images/brochure/STC.pdf

36. Matuschek et al, 2018
• The 5 commercial systems tested were:
4. Sensititre (Thermo Fisher Scientific, East
Grinstead, UK)
https://www.thermofisher.com/order/catalog/product/GN2F
COLISTIN AST STILL FOR RESEARCH USE ONLY

37. Matuschek et al, 2018
• The 5 commercial systems tested were:
5. UMIC (Biocentric, Bandol, France).
https://74da4a10-ac6d-4913-b1b1-
93846b360c26.filesusr.com/ugd/b49e33_02b6b266dd324065a712afe1e9326e3e.pdf

38. Matuschek et al, 2018
• While the 2 gradient tests used in their
investigation were:
MIC Test Strip (MTS,
Liofilchem, Roseto degli
Abruzzi, Italy).
Etest (bioMérieux, Marcy
l’Etoile, France)

39. Matuschek et al, 2018
• Seventy-five bacterial isolates were used in that
evaluation with varying levels of colistin
susceptibility, of which:
– P. aeruginosa (n=21),
– K. pneumoniae (n=18),
– E. coli (n=14), and
– Acinetobacter spp. (n=22, 16 of which A. baumannii)
• For the interpretation of colistin MIC values, the
EUCAST colistin breakpoints for
Enterobacteriaceae, P. aeruginosa, and
Acinetobacter spp. were used.

40. Matuschek et al, 2018

41. Chew et al., 2017
• In another study, Chew et al. compared the
performance of:
– Sensititre,
– MicroScan (Beckman Coulter, CA, USA),
– Vitek 2 system (bioMérieux, Marcy l’Etoile, France),
– Etest
• Against the reference standard broth
microdilution test on 76 isolates of carbapenem-
resistant mcr-1-positive Enterobacteriaceae.
Chew et al., Journal of Clinical Microbiology. 2017

42. Chew et al., 2017
• The EUCAST colistin breakpoints were used for
interpretation of colistin MIC values

43. Chew et al., 2017
*NA couldn’t be determined due to a narrow MIC range.

44. Commercial BMD
• To date, none of the commercial BMD systems
is FDA approved.
– Lack of FDA clinical colistin breakpoints is the
main reason for the absence of FDA approved
commercial BMD systems.
• More studies are very crucial to be able to
draw a conclusion on the overall performance
of these commercial systems.

45. COLISTIN BROTH DISK ELUTION
METHOD

47. Colistin Broth Disk Elution Method
• A recently published paper (Jan/2019) by
Simner et al. evaluated colistin broth disk
elution method against commercial BMD
(MicroScan and Sensititre GNX2F panels) and
broth macrodilution methods on Gram-
negative Bacilli isolates (n=172).

48. Colistin Broth Disk Elution Method
• The colistin broth disk elution method does not
require working with non-affordable equipment
or laborious techniques as the case with the
reference standard BMD.
• It has a similar principle that was applied for
anaerobes antimicrobial susceptibility testing in
1973, where known concentrations of multiple
antimicrobial disks were eluted in a different
volume of broth, to get the standard doubling
dilutions to measure the MICs.

50. Colistin Broth Disk Elution Method
• MIC values were estimated using both the
CLSI breakpoints (for A. baumannii and P.
aeruginosa) and ECV values (for
Enterobacteriaceae).

51. Colistin Broth Disk Elution Method
• The overall categorical and essential
agreement showed excellent performance of
this method when compared to standard BMD
methods (98 % and 99 % respectively).
• No MEs were reported, while overall VMEs
were at 8 %.

52. Colistin Broth Disk Elution Method
Conclusion
• Therefore, colistin broth disk elution method
is a very promising technique for colistin
antimicrobial susceptibility testing evaluation.
• Although, it is an accurate and practical
method, additional evaluation studies are
required to determine its overall performance.

53. CONCLUSION

54. Conclusion
Colistin susceptibility testing remains a subject
of continuing debate.
To this day, broth microdilution is the only
method endorsed and recommended by both
CLSI and EUCAST.
Various commercial BMD systems and novel
methods, such as the colistin broth disk elution
method, are nowadays available but further studies
are required for evaluation of their peroformance.

55. Acknowledgment
• The author is thankful to Dr. Fatimah Al Turki
(Microbiology Specialist, King Fahd Hospital of
The University, Dammam, Saudi Arabia) for
her valuable comments that greatly improved
the manuscript.

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57. THANK YOU