bsac susceptibility testing residential workshop...
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BSAC Susceptibility Testing Residential Workshop
Glycopeptide Resistance in Enterococci and Staphylococci
Gram positive resistance
Mandy Wootton
BSAC Susceptibility Testing Residential Workshop
MRSA
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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
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MRSA
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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.
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Usage drives mutation
Mupirocin usage and resistance in one Ward
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Usage Resistance
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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
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Disc BPs found in Topical agents tab
Macrolide resistance in Staphylococci /Streptococci
Protein synthesistermination
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
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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
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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”.
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Reporting if D-test negative:
Report clindamycin as tested using clinical breakpoints
VRE Prevalence
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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
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Glycopeptide - mode of action
vanAvanBvanDvanM
vanCvanEvanGvanLvanN
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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
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VRE – Types of Glycopeptide Resistance
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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
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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
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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
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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
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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
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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)
Protein synthesistermination
Glycopeptide Resistance in Staphylococci
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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.
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Disc diffusion does not detect R
Disc susceptibility testing
GRSA
Detected by disc
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MIC Determination – Microbroth dilution
MIC <0.25 0.5 1 2 4 8 16
GSSAhGISA
GISA
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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
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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
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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
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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
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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
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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 < 0.9hGISA 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
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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
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Any Questions?
BSAC Susceptibility Testing Workshop 2019