BSAC Susceptibility Testing Residential Workshop...

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 2019

Prevalence in UK : ~7%

2013: 13.7% 2014: 11.3% 2015: 10.8% 2016: 6.7% 2017: 6.9%

In Top Ten of pathogens causing blood stream infections

Associated with significant mortality & morbidity

Colonisation / screening

EARSNet 2017 MRSA from

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

– 0.45% of MRSA

mecI mecR1 mecA

Mupirocin resistance in S. aureus

Protein synthesistermination

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 –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≤ = 30R< = 18


Disc BPs found in Topical agents tab

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 mechanismSR

Type 2: Organism resistant to erythromycin and clindamycin

(constitutive MLSB) RR

Type 1: Organism susceptible to both erythromycin and

clindamycinSS

InterpretationCLINDAERYTH

Ery 15 ug Clin 2 ug

Disc screenD-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”.


Reporting if D-test negative:

Report clindamycin as tested using clinical breakpoints

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 prevalence VRE. faecium– 2013: 23.3%– 2014: 21.3%– 2015: 17%– 2016: 17%– 2017: 25.8%

Colonisation implicated

Trend ↓

Trend ↑

Gram +ve Cell Wall Biosynthesis


Glycopeptide - mode of action

vanAvanBvanDvanM

vanCvanEvanGvanLvanN


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 resistanceAcquired 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 locationPlasmid (Chr)

Plasmid (Chr)

Chr(plasmid)

Chr(plasmid)

Chr Chr ? Chr Chr

Precursors end Lac Lac Lac Lac Ser Ser Ser Ser Ser


Cattoir & Leclerc, JAC 2013

VRE – PhenotypesVAN 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 phenotypevanD genotype with vanB phenotype

Species ID important

E. gallinarum - vanA / vanBE. raffinosus - vanA

But not always


VRE 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/Bresistance

Read zone with plate held up to light

Look for small colonies or diffuse edge.

VRE Susceptibility Testing


EUCAST Warnings July 2018

MIC determination by gradient strips

Etest and MIC Test strip implicated

Isolates with vanB, low level R

Under estimate R

Significant numbers with MIC≤4mg/L by gradient strip have MICs 8 - ≥32mg/L by BMD

Linezolid resistance in Enterococcus faecalis

Refer any isolates:

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)

2012 – cfr gene – (plasmid mediated 23S RNA methyltransferase)

2016 – optrA gene (plasmid mediated transporter)

2018 – poxtA gene (plasmid mediated transporter)


Glycopeptide Resistance in Staphylococci


Glycopeptide Resistant S. aureus (GRSA)– vanA gene present (<30 World-wide)

– VAN MIC >32mg/L

Glycopeptide Intermediate S. aureus (GISA)– Homogeneous resistance (~120 World-wide)

– Resistance mechanism unknown

– VAN MIC ~4/8mg/L

Heterogeneous Glycopeptide Intermediate S. aureus (hGISA)– Heterogeneous resistance (0.25-0.9% MRSA)

– Resistance mechanism unknown

– VAN MIC ~1.5/4mg/L

– Precursor to GISA

EUCAST BreakpointsBorderline result of 2mg/L may have impaired clinical response

Susceptibility to dalbavancin & Oritavancin can be inferred from vancomycin.


Disc diffusion does not detect R

Disc susceptibility testing

GRSA

Detected by disc


MIC Determination – Microbroth dilution

MIC <0.25 0.5 1 2 4 8 16

GSSAhGISA

GISA


EUCAST recommend MBD‒ 105 inoculum

‒ Mueller Hinton Broth

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

Study 114 isolates (59 MRSA/GSSA, 47 hGISA, 8 GISA

>=-2 -2 -1 0 +1 +2 >+2

Etest 1 0 27 73 13 0 0

MICE 0 1 3 75 35 0 0

MIC Test 1 5 60 48 0 0 0

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/L1) 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

Macro gradient strip 100uL of 2 McFarland inoculum

Brain Heart Infusion Agar

48h incubation at 35±1°C in air

Interpretive criteria:

GISA or hGISA if:

VA or TP 8mg/L

OR

TP 12mg/L1) 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 at 48hDetects most hGISA2% false positives

GSSA hGISA GISA

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

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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 < 0.9hGISA 0.9 - 1.29GISA 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?


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