difficult or tricky antibiotic resistance phenotypes to ... · 201 s. aureus (51 mrsa) 4 mrsa...
TRANSCRIPT
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Difficult or Tricky Antibiotic Resistance Phenotypes to Recognize
Tan Thean Yen
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Contents
Gram-positive
Staphylococcus aureus with penicillinase production
Vancomycin resistant Enterococci (VRE)
Staphylococcus aureus with reduced susceptibility to glycopeptides (hVISA, VISA, VRSA)
Gram-negative
polymyxin testing
Carbapenemases (IMP, VIM, KPC, and OXA-48)
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Staphylococcus aureusPenicillinase production
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penicillin susceptibility
CLSI
10U disc
R ≤ 28 mm [R ≥ 0.25 mg/L]
S ≥ 29 mm [S ≤ 0.12 mg/L]
EUCAST
1U disc
R < 26 mm [R > 0.12 mg/L]
S ≥ 26 mm [S ≤ 0.12 mg/L]
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penicillin susceptibility
CLSI10U disc
R ≤ 28 mm [R ≥ 0.25 mg/L]
S ≥ 29 mm [S ≤ 0.12 mg/L]
EUCAST
1U disc
R < 26 mm [R > 0.12 mg/L]
S ≥ 26 mm [S ≤ 0.12 mg/L]
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penicillin susceptibility
CLSI
10U disc
R ≤ 28 mm [R ≥ 0.25 mg/L]
S ≥ 29 mm [S ≤ 0.12 mg/L]
EUCAST1U disc
R < 26 mm [R > 0.12 mg/L]
S ≥ 26 mm [S ≤ 0.12 mg/L]
For S. aureus, disk diffusion is more reliable than MIC determination for detection of penicillinase producers, provided the zone diameter is measured AND the zone edge closely inspectedExamine the zone edge with transmitted light (plate held up to light).If the zone diameter is <26 mm, then report resistant. If the zone diameter is ≥26 mm AND the zone edge is sharp, then report resistant. If not sharp, then report susceptible and if uncertain, then report resistant.Chromogenic cephalosporin-based beta-lactamase tests do not reliably detect staphylococcal penicillinase.
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Examples of inhibition zones for Staphylococcus aureus with benzylpenicillin.
a) Fuzzy zone edge and zone diameter ≥ 26 mm. Report susceptible.b) Sharp zone edge and zone diameter ≥ 26 mm. Report resistant.
Transmitted light, dark background, no magnifying glass
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Papanicolas LE, Bell JM, Bastian I. Performance of phenotypic tests for detection of penicillinase in Staphylococcus aureus isolates from Australia. Journal Of Clinical Microbiology. 2014 Apr 1;52(4):1136-8.
Conclusions:Cefinase disc not reliableCLSI zone edge performed better, but not 100%
CLSI
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Papanicolas LE, Bell JM, Bastian I. Performance of phenotypic tests for detection of penicillinase in Staphylococcus aureus isolates from Australia. Journal Of Clinical Microbiology. 2014 Apr 1;52(4):1136-8.
Conclusions:Cefinase disc not reliableCLSI zone edge performed better, but not 100%EUCAST zone edge performed the best
EUCAST
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Hombach M, Weissert C, Senn MM, Zbinden R. Comparison of phenotypic methods for the detection of penicillinase in Staphylococcus aureusand proposal of a practical diagnostic approach. Journal of Antimicrobial Chemotherapy. 2016 Dec 20;72(4):1089-93.
Conclusions:EUCAST zone edge performed quite wellInter-observer variability
Note variability in sensitivity,depending on observer
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Hombach M, Weissert C, Senn MM, Zbinden R. Comparison of phenotypic methods for the detection of penicillinase in Staphylococcus aureusand proposal of a practical diagnostic approach. Journal of Antimicrobial Chemotherapy. 2016 Dec 20;72(4):1089-93.
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Staphylococcus aureusVancomycin resistance
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vancomycin & S. aureus
Susceptible Intermediate Resistant
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MRSA / VISA / VRSA
hVISA – heterogenously vancomycin intermediate S. aureus
VISA VRSA
Susceptible Intermediate Resistant
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hVISA and MIC
Howden BP, et al.
Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate
strains: resistance mechanisms, laboratory detection, and clinical implications. Clinical Microbiology Reviews. 2010 Jan 1;23(1):99-139.
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Vancomycin-intermediate S. aureus (VISA)First described in Japan in 1997
Intermediate resistant (MIC 4-8 mg/L)
Hetero-intermediate resistant (MIC <4 mg/L)
Increased production of cell wall precursors and PBP 2’
Reduced cross linking in cell wall (less target for vancomycin)
Lower growth rates and thicker cell walls (vancomycin sequestration)
Prolonged exposure to vancomycin
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MRSA VISA
Micky LEONG (Ms) :: Laboratory Technologist, Electron Microscopy Unit YLLSOM
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Vancomycin intermediate Staphylococcus aureus
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Lab detection of VISA
Not detected by routine disk or automated methods
Etest (consistently one twofold dilution higher)
Broth microdilution
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What is the vancomycin MIC?
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What is the vancomycin MIC?
Incubate full 24 hours.
Inspect closely for micro-colonies at the intersection edge.
Incubate 48 hours if high clinical suspicion.
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EUCASTMIC breakpoint (mg/L) Zone diameter breakpoint
(mm)
S ≤ R > S ≥ R <
Vancomycin 2 2 - -
CLSIMIC breakpoint (mg/L) Zone diameter breakpoint
(mm)
S ≤ I R ≥ S ≥ I R ≤
Vancomycin 2 4-8 16 - - -
Glycopeptide MICs are method dependent and should be determined by broth microdilution (reference ISO 20776). S. aureus with vancomycin MIC values of 2 mg/L are on the border of the wild type distribution and there may be an impaired clinical response. The resistant breakpoint has been reduced to 2 mg/L to avoid reporting ""GISA"" isolates intermediate as serious infections with ""GISA"" isolates are not treatable with increased doses of vancomycin or teicoplanin. Disk diffusion is unreliable and cannot distinguish between wild type isolates and those with non-vanA-mediated glycopeptide resistance. "
MIC tests should be performed to determine the susceptibility of all isolates of staphylococci tovancomycin. The disk test does not differentiate vancomycin-susceptible isolates of S. aureus fromvancomycin-intermediate isolates…The vancomycin 30-μg disk test detects S. aureus isolates containing the vanA vancomycin resistance gene (VRSA). Such isolates will show no zone of inhibition around thedisk (zone = 6 mm)…
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How does your lab measure the MIC?
Etest
Automated MIC
Broth dilution
0.5 - 1 dilution higher than broth microdilution (gold standard)
1-2 dilutions lower than gold standard
Gold standard? correlate with clinical outcome?
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Vitek versus Etest (92 isolates)
0
10
20
30
40
50
60
70
0.5 1 2
Total
0
10
20
30
40
50
60
70
0.5 1 2 4
Total
Vitek Etest
Dr Tan TY, Dept of Lab Medicine, Changi General Hospital
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Vitek and vancomycin
Study 1
201 S. aureus (51 MRSA)
4 MRSA reported as OX=S
5 MSSA reported as OX=R
6 VISA reported as VA=S
Study 2
417 MRSA isolates
Vitek2® MIC results were 1–2 dilutions lower than BMD for both low and high BMD MICs
Morgado PG, et al.. VITEK® 2 cannot identify vancomycin intermediate isolates: missing the opportunity for Staphylococcus aureus therapy.
Journal of Hospital Infection. 2017 Jul 19.
Van Hal SJ, et al. Methicillin-resistant Staphylococcus aureus vancomycin susceptibility testing: methodology correlations, temporal trends and
clonal patterns. Journal Of Antimicrobial Chemotherapy. 2011 Jul 12;66(10):2284-7.
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(Hetero) hVISA
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Lab detection of hVISA
Population analysis
Macrodilution Etest (not true MIC)
Etest GRD
MHA5T screening plate (5 μg/ml teicoplanin)
hVISA may predict potential failure of daptomycin therapy
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Howden BP, et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous
vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clinical microbiology reviews. 2010 Jan
1;23(1):99-139.
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Howden, B. P. et al. 2010. Clin. Microbiol. Rev. 23(1):99-139
FIG. 8. Examples of Etest methodology used to detect hVISA
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simpler method
10-µl spots of a 0.5 McFarland suspension in saline
inoculated onto vancomycin-BHI plates(3 mg/L and 4 mg/L)
incubation at 35°C for 48 hours
colonial spots counted
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Khatib R, et al. Screening for Intermediately Vancomycin-Susceptible and Vancomycin-Heteroresistant Staphylococcus aureus by Use of Vancomycin-Supplemented Brain Heart Infusion Agar Biplates: Defining Growth Interpretation Criteria Based on Gold Standard Confirmation. Journal of clinical microbiology. 2015 Nov 1;53(11):3543-6.
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Agar Colonies Interpretation
(BHI-Va4) 0 VSSA
1 VSSA / hVISA
2-15 hVISA / VISA
≥16 VISA
(BHI-Va3) 0-2 VSSA
≥2 VISA / hVISA
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Vancomycin-resistant S. aureus (VRSA)In-vitro transfer of VanA in 1992
Michigan (June 2002), Pennsylvania (Sept 2002)
MIC 1024 mg/L, 32 mg/L
VanA positive
Plasmid-mediated
Detected by disk diffusion but may not be detected by automated methods
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Tenover, F. C. et al. 2004. Antimicrob. Agents Chemother. 48(1):275-280
FIG. 1. Disk diffusion and Etest analysis of the PA-VRSA isolate on Mueller-Hinton agar
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Enterococcus faecium / faecalisVancomycin resistance
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Vancomycin-resistant Enterococcus
VanA VanB VanC
Vanc MIC
mg/L
64->1000
Resistant
4-1024
Resistant
2-32
Intermediate
Teic MIC
Mg/L
16-512
Resistant
0.5
Susceptible
0.5
Susceptible
Species E. Faecium
E. faecalis
E. Faecium
E. faecalis
E. Gallinarum
E. Casseiflavus
E. flavescens
Genetic
determinant
Acquired Acquired Intrinsic
Transferable Yes Yes No
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VSE
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Van A VRE
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Van B VRE
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VRE
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(A) Cultures with sharp zone edges and zone diameters of ≥12 mm should be reported as susceptible. (B to D) Cultures with fuzzy zone edges (C and D) or colonies within the zone (B) should be reported as resistant, even if the zone diameter is ≥12 mm.
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VRE Lab Detection
Disk diffusionincubate full 24 h, view with transmitted light for hazy zones
MIC methods
Screening plates(vancomycin 6 mg/L)
Multiplex PCR to detect vanA, vanB
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Identify all VRE to species level
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vanC motile E. gallinarum
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EnterobacteriaceaeCarbapenemase producers
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Carbapenemases
Class A
KPC
Class B
IMP, VIM, NDM
Class C
Class D
OXA-48
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EUCASTMIC breakpoint (mg/L) Zone diameter breakpoint
(mm)
S ≤ R > S ≥ R <
Ertapenem 0.5 1 25 22
Imipenem 2 8 22 16
Meropenem 2 8 22 16
CLSIMIC breakpoint (mg/L) Zone diameter breakpoint
(mm)
S ≤ I R ≥ S ≥ I R ≤
Ertapenem 0.5 1 2 22 19-21 18
Imipenem 1 2 4 23 20-22 19
Meropenem 1 2 4 23 20-22 19
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Carbapenemases
Clinical antimicrobial susceptibility?
Infection control purposes
Epidemiological / public health
detection
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CP-CRE vs non-CP-CRE
83 bacteraemia episodes
37 CP-CRE
46 non-CP-CRE
CP Enzymes
KPC n=32 (92%)
NDM n=2 (5%)
OXA n=1 (3%)
Patients
well balanced for demographic & pre-existing conditions
Pitt bacteraemia score similar
CP-CRE less likely to receive active empiric abx therapy (ns)
Tamma PD, Goodman KE, Harris AD, Tekle T, Roberts A, Taiwo A, Simner PJ. Comparing the Outcomes of Patients With Carbapenemase-Producing and Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae Bacteremia. Clinical Infectious Diseases. 2017 Feb 1;64(3):257-64.
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Antibiotics suceptibility
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CP-CRE vs non-CP-CRE
Mortality
Overall 22%
CP-CRE n=12 (32%)
Non-CP-CRE n=6 (13%)
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CP-CRE vs non-CP-CREMultivariate
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Karlowsky JA, et al. In Vitro Activity of Imipenem against Carbapenemase-Positive Enterobacteriaceae Isolates Collected by the SMART Global Surveillance Program from 2008 to 2014. Journal of Clinical Microbiology. 2017 Jun 1;55(6):1638-49.
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can use different breakpoints for screening for carbapenemases
The EUCAST guideline on detection of resistance mechanisms v 2.0 (2017-07-11). Available from:http://www.eucast.org/resistance_mechanisms/
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Maurer FP, et al. Evaluation of carbapenemase screening and confirmation tests with Enterobacteriaceae and development of a practical diagnostic algorithm. Journal of clinical microbiology. 2015 Jan 1;53(1):95-104.
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Phenotype
boronic acid synergy test using 10µg
ertapenem disks
metallo-β-lactamase (MβL) Etest(imipenem & imipenem/EDTA)
modified Hodge test
Inhibition testing
Inactivation carbapenem inactivation method (CIM)
modified carbapenem inactivation method
(mCIM)
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Tamma PD, Opene BN, Gluck A, Chambers KK, Carroll KC, Simner PJ. A Comparison of Eleven Phenotypic Assays for the Accurate Detection of
Carbapenemase-Producing Enterobacteriaceae. Journal of Clinical Microbiology. 2017 Jan 11:JCM-02338.
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Tamma PD, Opene BN, Gluck A, Chambers KK, Carroll KC, Simner PJ. A Comparison of Eleven Phenotypic Assays for the Accurate Detection of
Carbapenemase-Producing Enterobacteriaceae. Journal of Clinical Microbiology. 2017 Jan 11:JCM-02338.
sensitivity 98%
specificity 99%
SME (88%)
OXA-48 (93%)
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Tamma PD, Opene BN, Gluck A, Chambers KK, Carroll KC, Simner PJ. A Comparison of Eleven Phenotypic Assays for the Accurate Detection of
Carbapenemase-Producing Enterobacteriaceae. Journal of Clinical Microbiology. 2017 Jan 11:JCM-02338.
sensitivity 98-100%
specificity 96-100%
NDM (97%)
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Tamma PD, Opene BN, Gluck A, Chambers KK, Carroll KC, Simner PJ. A Comparison of Eleven Phenotypic Assays for the Accurate Detection of
Carbapenemase-Producing Enterobacteriaceae. Journal of Clinical Microbiology. 2017 Jan 11:JCM-02338.
sensitivity 98%
specificity 99%
IMP (83%)
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So, which test to use?
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EnterobacteriaceaePolymyxin susceptibility
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polymyxin
intrinsically difficult to test
• poor diffusion in agar
• inherent cationic properties
• hetero-resistance in some species
• lack of reliable reference method
Poirel L, Jayol A, Nordmann P. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes.
Clinical Microbiology Reviews. 2017 Apr 1;30(2):557-96.
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broth dilution current reference standard
unresolved issues
cation concentrations, formulation of colistinsulfate, binding to materials, P-80 surfactant
agar dilution possible, but not reference
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disc susceptibility testing
very small zones of inhibition
CLSI 10µg
EUCAST 50µg
does not reliably detect resistance
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Etest
varying results
may over- and under-estimate resistance
Chew KL, La M-V, Lin RTP, Teo JWP. 2017. Colistin and polymyxin B susceptibility testing for carbapenem-resistant and mcr-positive Enterobacteriaceae:
comparison of Sensititre, MicroScan, Vitek 2, and Etest with broth microdilution. J Clin Microbiol 55:2609 – 2616.
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Etest
Evaluation of five commercial MIC methods for colistin antimicrobial susceptibility testing for Gram-negative bacteria. Available from:
http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Warnings/Matuschek_colistin_ECCMID_2017.pdf
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Etest
Dafopoulou K, et al. Comparative evaluation of colistin susceptibility testing methods among carbapenem-nonsusceptible Klebsiella pneumoniae and
Acinetobacter baumannii clinical isolates. Antimicrobial agents and chemotherapy. 2015 Aug 1;59(8):4625-30.
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Etest
hetero-resistance
Lo-Ten-Foe JR, et al. Comparative evaluation of the VITEK 2, disk diffusion, Etest, broth microdilution, and agar dilution susceptibility testing methods for
colistin in clinical isolates, including heteroresistant Enterobacter cloacae and Acinetobacter baumannii strains. Antimicrobial Agents And Chemotherapy.
2007 Oct 1;51(10):3726-30.
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Automated methods
Chew KL, La M-V, Lin RTP, Teo JWP. 2017. Colistin and polymyxin B susceptibility testing for carbapenem-resistant and mcr-positive nterobacteriaceae:
comparison of Sensititre, MicroScan, Vitek 2, and Etest with broth microdilution. J Clin Microbiol 55:2609 – 2616.
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Automated methods
Chew KL, La M-V, Lin RTP, Teo JWP. 2017. Colistin and polymyxin B susceptibility testing for carbapenem-resistant and mcr-positive nterobacteriaceae:
comparison of Sensititre, MicroScan, Vitek 2, and Etest with broth microdilution. J Clin Microbiol 55:2609 – 2616.
We could not systematically evaluate semi-automated colistinmethods but by sending isolates with MIC values in the non-susceptible range to colleagues around the world we have disclosed the frequent occurrence of Very Major Errors.Users of semi-automated devices should apply rigorous QC (see below) and check with the manufacturer whether or not they are confident that their method for colistin AST gives correct results.Quality control of colistin must be performed with both a susceptible QC strain (E. coli ATCC 25922 or P. aeruginosa ATCC 27853) and the colistin resistant E. coli NCTC 13846 (mcr-1 positive). For E. coli NCTC 13846, the colistin MIC target value is 4 mg/L and should only occasionally be 2 or 8 mg/L.
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Nordmann P, Jayol A, Poirel L.
Rapid detection of polymyxin resistance in Enterobacteriaceae. Emerging infectious diseases. 2016 Jun;22(6):1038.
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…so…
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“Some things in life are too complicated to explain in any language.
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susceptibility testing is simple..
..
until the bugs get complicated
..
…
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Douglas Adams.
The Hitchhikers Guide To the Galaxy.
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Choose clinically important issues.
In your area.
Read the literature.
Assess: Time.
Money.Speed of results.
Then:
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*
* with apologies to Nike
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Tan Thean Yen
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AmpC in Gram-negative bacilli
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Class C beta-lactamasesGene normally found in most Gram negatives except Klebsiella and Salmonella
Cephalosporinases
Not inhibited by clavulanate, tazobactam (except M. morgannii)
Resistant to cefoxitin
Cefepime not generally affected
Chromosomal
Generally expressed at low level but are inducible
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ESCHAPPM
Enterobacter cloacae/aerogenes
Serratia marcescens
Citrobacter freundiiHafnia alveiAeromonas hydrophila/caviaeProvidencia stuartii/rettgeri Morganella morganii
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Induction vs Stable Derepression
Induction
transient switching on of -lactamase synthesis in response to an inducer (cefoxitin, imipenem)
Stable Derepression
permanent hyperproduction of the -lactamase independent of an inducer
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Chromosomal AmpC in Enterobacterspp.
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Ceftriaxone
Ceftoxitin
Ceftriaxone
Cefoxitin
Augmentin
Aztreonam
K. Pneumoniae
Would you treat this patient with
ceftriaxone?
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Ceftriaxone
Cefoxitin
De-repressed mutants
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De-repressed
mutantOriginal strain
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Original
strain
De-repressed
mutant
>32 mg/L-
-1.5 mg/L1mg/L susceptible-
pAmpC DHA-1
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DHA in Klebsiella pneumoniae
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CLSI(8) Enterobacter, Citrobacter, and Serratia may develop resistance during prolonged therapy with third-generation cephalosporins as a result of derepression of AmpC β-lactamase. Therefore, isolates that are initially susceptible may become resistant within three to four days after initiation of therapy. Testing of repeat isolates may be warranted.
Enterobacter spp., Citrobacter freundii, Serratia spp. and Morganella morganii. If susceptible in- vitro, the use of monotherapy of cefotaxime should be discouraged, owing to the risk of selection of resistance, or suppress the susceptibility testing result for this agent.
BSAC (EUCAST?)
Inducible plasmid AmpC (DHA) in K. pneumoniae?
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CDS method•E. cloacae, E. aerogenes, C. freundii, inducible pAmpC
–High frequency of derepressed mutants (10-5 to 10-6)
–Report R to all cephalosporins
–Test cefepime and carbapenems
–Do not attempt to test other β-lactams
•S. marcescens
–No derepressed mutants with ceftazidime, tazocin and aztreonam
–Derepressed mutants with other β-lactams including cefotaxime.
•H. alvei, P. stuartii, P. rettgeri and M. morganii
–Very low mutation rate (10-8)
–Test and report accordingly
http://web.med.unsw.edu.au/cdstest/