Difficult or Tricky Antibiotic Resistance Phenotypes to Recognize

Tan Thean Yen

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)

Staphylococcus aureusPenicillinase production

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]

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]

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.

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

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

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

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

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.

Staphylococcus aureusVancomycin resistance

vancomycin & S. aureus

Susceptible Intermediate Resistant

MRSA / VISA / VRSA

hVISA – heterogenously vancomycin intermediate S. aureus

VISA VRSA

Susceptible Intermediate Resistant

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.

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

MRSA VISA

Micky LEONG (Ms) :: Laboratory Technologist, Electron Microscopy Unit YLLSOM

Vancomycin intermediate Staphylococcus aureus

Lab detection of VISA

Not detected by routine disk or automated methods

Etest (consistently one twofold dilution higher)

Broth microdilution

What is the vancomycin MIC?

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.

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)…

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?

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

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.

(Hetero) hVISA

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

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.

Howden, B. P. et al. 2010. Clin. Microbiol. Rev. 23(1):99-139

FIG. 8. Examples of Etest methodology used to detect hVISA

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

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.

Agar Colonies Interpretation

(BHI-Va4) 0 VSSA

1 VSSA / hVISA

2-15 hVISA / VISA

≥16 VISA

(BHI-Va3) 0-2 VSSA

≥2 VISA / hVISA

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

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

Enterococcus faecium / faecalisVancomycin resistance

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

VSE

Van A VRE

Van B VRE

VRE

(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.

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

Identify all VRE to species level

vanC motile E. gallinarum

EnterobacteriaceaeCarbapenemase producers

Carbapenemases

Class A

KPC

Class B

IMP, VIM, NDM

Class C

Class D

OXA-48

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

Carbapenemases

Clinical antimicrobial susceptibility?

Infection control purposes

Epidemiological / public health

detection

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.

Antibiotics suceptibility

CP-CRE vs non-CP-CRE

Mortality

Overall 22%

CP-CRE n=12 (32%)

Non-CP-CRE n=6 (13%)

CP-CRE vs non-CP-CREMultivariate

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.

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/

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.

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)

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.

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%)

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%)

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%)

So, which test to use?

EnterobacteriaceaePolymyxin susceptibility

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.

broth dilution current reference standard

unresolved issues

cation concentrations, formulation of colistinsulfate, binding to materials, P-80 surfactant

agar dilution possible, but not reference

disc susceptibility testing

very small zones of inhibition

CLSI 10µg

EUCAST 50µg

does not reliably detect resistance

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.

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

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.

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.

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.

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.

Nordmann P, Jayol A, Poirel L.

Rapid detection of polymyxin resistance in Enterobacteriaceae. Emerging infectious diseases. 2016 Jun;22(6):1038.

…so…

“Some things in life are too complicated to explain in any language.

susceptibility testing is simple..

..

until the bugs get complicated

..

…

Douglas Adams.

The Hitchhikers Guide To the Galaxy.

Choose clinically important issues.

In your area.

Read the literature.

Assess: Time.

Money.Speed of results.

Then:

*

* with apologies to Nike

Tan Thean Yen

AmpC in Gram-negative bacilli

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

ESCHAPPM

Enterobacter cloacae/aerogenes

Serratia marcescens

Citrobacter freundiiHafnia alveiAeromonas hydrophila/caviaeProvidencia stuartii/rettgeri Morganella morganii

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

Chromosomal AmpC in Enterobacterspp.

Ceftriaxone

Ceftoxitin

Ceftriaxone

Cefoxitin

Augmentin

Aztreonam

K. Pneumoniae

Would you treat this patient with

ceftriaxone?

Ceftriaxone

Cefoxitin

De-repressed mutants

De-repressed

mutantOriginal strain

Original

strain

De-repressed

mutant

>32 mg/L-

-1.5 mg/L1mg/L susceptible-

pAmpC DHA-1

DHA in Klebsiella pneumoniae

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?

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/