BSAC Susceptibility Testing Residential Workshop Workshop · 2019-11-18 · GISA BSAC Susceptibility Testing Workshop 2016 105 inoculum Mueller Hinton Broth Incubation at 35±1°C

BSAC Susceptibility Testing Residential Workshop

Glycopeptide Resistance in Enterococci and Staphylococci

Gram positive resistance

Mandy Wootton

BSAC Susceptibility Testing Residential Workshop

MRSA

BSAC Susceptibility Testing Workshop 2016

Prevalence in UK : ~11%

In Top Ten of pathogens causing blood stream infections

Associated with significant mortality & morbidity

Colonisation / screening

MRSA


MRSA


Have an additional PBP2a / PBP2’

2011 mecC in bovine mastitis – Animals & humans all over Europe – Phenotypic testing OK – Chromogenic agar OK – some variability – NEG with PBP latex kits – Low expression of PBP2a – NEG with PCR primers – Automated systems: OXA S, FOX R – BSAC disc misses 29% of mecC

– 2-3% of MRSA

mecI mecR1 mecA

Mupirocin resistance in S. aureus

Protein synthesis termination

Mechanism of action: Binds to isoleucine (ileS) tRNA synthetase Low level resistance

– MIC 8-256mg/L

– Mutation in ileS gene

High level resistance

– MIC ≥512mg/L

– Acquisition of mupA gene; novel isoleucine tRNA synthetase.


Usage drives mutation

Mupirocin usage and resistance in one Ward

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Usage Resistance


Mupirocin resistance in S. aureus –

Detection of differentiation between low & high level resistance

EUCAST

Disc 200ug or MIC

Breakpoints relate to nasal decolonisation

Low level resistance (Intermediate)

– Short term suppression

– Long term eradication rate are low

S≤ = 30 R< = 18


Macrolide resistance in Staphylococci /Streptococci


Mechanism of action: Binds to 23S ribosomal subunit Target modification

– erm gene methylates 23S – Confers R to Macrolide-Lincosamides-

type B Streptogramins (MLSB) – Expression constitutive or inducible

Efflux – Msr(A) – ABC transporter – Confers R to 14 & 15-member ring

macrolides –type B Streptogramins (MS)

Antimicrobial inactivation – ereA/ereB – hydrolyses macrolides – lnuA – hydrolyses lincosamides


Macrolide resistance in Staphylococci /Streptococci - Detection

Type 3: May have inducible resistance (inducible MLSB) or

other mechanism S R

Type 2: Organism resistant to erythromycin and clindamycin

(constitutive MLSB) R R

Type 1: Organism susceptible to both erythromycin and

clindamycin S S

Interpretation CLINDA ERYTH

Ery 15 ug Clin 2 ug

Disc screen D-test

Placed 12-20mm apart

Streps: Placed 12-16mm apart


Macrolide resistance in Staphylococci /Streptococci - Reporting

Reporting if D-test positive:

Staphylococci & Streptococci report as clindamycin R – Add comment “Clindamycin may still be used for short-term

therapy of less serious skin & soft tissue infections as constitutive resistance is unlikely to develop during therapy”.

Pre 2015 EUCAST (v4) & 2014 BSAC (v13): – Staphylococci – report as R

– Streptococci – report as S


VRE Prevalence


3rd most common cause of nosocomial blood stream infections

Account for 10-25 % of all blood stream infections

E. faecium & E. faecalis predominate

UK & Europe prevalence – E. fcm / 10-25% VRE, E. fcl / 1-3% VRE

Colonisation implicated

Gram +ve Cell Wall Biosynthesis


Glycopeptide - mode of action

vanA vanB vanD vanM

vanC vanE vanG vanL vanN


VRE - Mechanism of Glycopeptide resistance

vanA / vanB vanS vanR vanH vanX vanY vanZ

Ligase – adds lactate or serine to D-ala

Membrane protein – detects presence of Vanc

Turns on all other van genes

Synthesises D-lactate

DD dipeptidase – cleaves D-ala – D-ala

Cleaves D-ala from pentapeptide

Associated with teicoplanin R – mechanism unclear


VRE – Types of Glycopeptide Resistance


Acquired resistance

Species – E. faecalis

– E.faecium

Genes – vanA, vanB, vanD, vanE, vanG,

vanL, vanM, vanN

Transposable elements – Outbreak associated

Intrinsic resistance

Species – E. gallinarum

– E.flavenscens

– E. casseliflavus

Genes – vanC

Chromosomal – Not outbreak associated

VRE – Level of resistance Acquired resistance

Intrinsic resistance

High level Variable Moderate Low level Low level

vanA vanM vanB vanD vanE vanG vanL vanN vanC1,2,3

Vancomycin susceptibility

R R R-r R r r r r r

Teicoplanin susceptibility

R R S R-R S S S S S

Transferability + + + - - + - + -

Main enterococcal spp

Fcm/Fcl/other

Fcm Fcm/Fcl Fcm/Fcl Fcl Fcl Fcl Fcm Gal/Cas

Expression I ? I C I/C I I C C/I

Genetic location Plasmid (Chr)

Plasmid (Chr)

Chr (plasmid)

Chr (plasmid)

Chr Chr ? Chr Chr

Precursors end Lac Lac Lac Lac Ser Ser Ser Ser Ser


VRE – Phenotypes VAN TEIC

vanA >128 >16

vanB 16-64 1

vanC/D 2-32 0.5-1

vanD (E. faecium) 64-128 4-8

vanA >256 4

vanA genotype with vanB phenotype vanD genotype with vanB phenotype

Species ID important

E. gallinarum - vanA / vanB E. raffinosus - vanA

But not always


Susceptibility Testing

Glycopeptides

MIC breakpoint (mg/L) Disk

content (µg)

Zone diameter breakpoint (mm)

S ≤ R > S ≥ R <

Teicoplanin 2 2 30 16 16

Vancomycin 4 4 5 12 12


Disc diffusion - EUCAST 5ug vancomycin disc

30ug teicoplanin disc

0.5 McFarland inoculum on MHA

Incubation at 35±1°C in air for 24 hours – longer incubation time due to inducible nature of vanA/B resistance

Look for small colonies or diffuse edge.

Linezolid resistance in Enterococcus faecalis

OptrA gene – plasmid mediated transporter

Resistance alert 2012 - cfr gene

Cfr gene – plasmid mediated 23S RNA methlytransferase

Any isolates R by disc (<19mm) or MIC (>4mg/L) should be referred

Plasmid mediated so transferable


Resistance rare <1%

Most commonly caused by mutation (G2576T)

Resistance alert in June 2016 – optrA gene


Glycopeptide Resistance in Staphylococci


Glycopeptide Resistant S. aureus (GRSA) – vanA gene present

– VAN MIC >32mg/L

Glycopeptide Intermediate S. aureus (GISA) – Homogeneous resistance

– Resistance mechanism unknown

– VAN MIC ~4/8mg/L

Heterogeneous Glycopeptide Intermediate S. aureus (hGISA) – Heterogeneous resistance

– Resistance mechanism unknown

– VAN MIC ~1.5/4mg/L

– Precursor to GISA

– <30 World wide

– ~120 World wide

– Between 0.25-0.9%

EUCAST Breakpoints Borderline result of 2mg/L may

have impaired clinical response

Method dependant; perform

microbroth dilution


Glycopeptides1 MIC

breakpoint

(mg/L)

Disk

conten

t (µg)

Zone

diameter

breakpoint

(mm)

Notes

Numbers for comments on MIC breakpoints

Letters for comments on disk diffusion

S ≤ R > S ≥ R <

Teicoplanin, S. aureus 2 2 NoteA Note

A

1.

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 MIC 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.

2. For telavancin MIC determination, the medium must be supplemented

with polysorbate-80 to a final concentration of 0.002%.

3. MRSA isolates susceptible to vancomycin can be reported susceptible to

telavancin.

A. Disk diffusion is unreliable and cannot distinguish between wild type

isolates and those with non-vanA-mediated glycopepetide resistance.

Teicoplanin, Coagulase-negative

staphylococci

4 4 NoteA Note

A

Telavancin, MRSA 0.1252,

3

0.1252,

3

NoteA Note

A

Vancomycin, S. aureus 2 2 NoteA Note

A

Vancomycin, Coagulase-negative

staphylococci

4 4 NoteA Note

A

http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Rationale_documents/Teicoplanin_rationale_2.1.pdf


http://mic.eucast.org/SearchController/search.jsp?action=performSearch&BeginIndex=0&Micdif=mic&NumberIndex=50&Antib=54&Specium=-1









http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Rationale_documents/Vancomycin_rationale_2.1.pdf












Disc susceptibility testing

GRSA

Detected by disc


MIC Determination – Microbroth dilution

MIC <0.25 0.5 1 2 4 8 16

GSSA hGISA

GISA


105 inoculum

Mueller Hinton Broth

Incubation at 35±1°C in air for 18-20 hours

Ab MIC

method

GEOMean (mg/L) Range (mg/L) % resistance Sn Sp

GSSA hGISA GISA GSSA hGISA GISA GSSA hGISA GISA

VAN MBD 0.7 1.9 3.1 0.5-2 1-2 2-8 0 0 50 7.7 100

Gradient 0.5 1.3 2.5 0.25-1 1-2 1.5-4 0 0 50 7.7 100

TEIC MBD 0.4 2 4 0.25-1 1-8 2-8 0 27.3 62.5 32.7 100

Gradient 0.4 2.1 4.3 0.19-1.5 1-4 1.5-12 0 40.8 62.5 44.2 100

MIC Determination – Automated methods

Tenover et al 2010


GRD strip 0.5 McFarland inoculum

Mueller Hinton + sheep blood

48h incubation at 35±1°C in air – Reading at 24h and 48h

Interpretive criteria:

GISA or hGISA if VA or TP 8mg/L 1) GISA if GRD+ and standard VA MIC 4mg/L

2) hGISA if GRD+ and standard VA MIC <4mg/L

Cannot use to determine MIC

Detects all GISA, majority at 24h Detects most hGISA and GISA 2% false positives


Phenotype No + at 24hr

No + at 48hr

% correctly classified

GSSA (n=50) 0 1 98

hGISA (n=51) 30 41 80.4

GISA (n=26) 24 26 100

Screening Agars

Mueller Hinton Agar + 5mg/L Teicoplanin (MHA5T) – 10ul of 0.5 McF inoculum – 48h Incubation at 35-37°C in air – Interpretive criteria: hGISA or GISA if ≥2 cols after 48hr


Method % GSSA

correctly classified

% hGISA correctly classified

% GISA correctly classified

MHA5T 97 78.7 93.8

MHA MHA5T

Population Analysis Profile – Area Under Curve 1

“Gold Standard”

2 plates of BHIA + 0, 0.5, 1, 2, 2.5 & 4mg/L VA

Overnight cultures of test organism and Mu3 in TSB - diluted 1:10-3, 1:10-6

Incubation at 35-37°C in air for 48hrs

106 / 103 106 / 103 106 / 103 103 / N 103 / N 103 / N


PAP-AUC 2

PAP of Mu 3 (hGISA) , Mu50(GISA), GSSA and OxfordStaphylococcus aureus

10 2

10 3

10 4

10 5

10 6

10 7

10 8

10 9

10 1 0

Mu 3 (hGISA)

Mu50 (GISA)

NCTC 6571 (GSSA)

GSSA

0.5 1 2 2.5 4 8

Vancomycin concentration mg/L

Via

ble

co

un

t L

og

cfu

/ml

• Log viable count vs. VA concn graph

• Ratio = AUC of test isolate/AUC of Mu3

• PAP-AUC ratio criteria:

GSSA < 0.9 hGISA 0.9 - 1.29 GISA 1.3

Observation number

PA

P-A

UC

0 1 2 3 40.0

0.5

1.0

1.5hVISA

VISA

VSSA


hGISA/GISA summary

Do not disc test, even though GRSA detected

MIC determination is best BUT clinical need should drive extra work

Automated methods; beware the risks

Screening agars for large numbers

Confirmatory tests / reference laboratory


Any Questions?


Documents

BSAC Susceptibility Testing Residential Workshop Workshop · 2019-11-18 · GISA BSAC Susceptibility Testing Workshop 2016 105 inoculum Mueller Hinton Broth Incubation at 35±1°C