BSAC Susceptibility Testing Testing Residential Workshop Residential Workshop Gram positive resistance Glycopeptide Resistance in Enterococci and Staphylococci Mandy Wootton
MRSA 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 meci mecr1 meca 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
Mupirocin resistance in S. aureus Low level resistance Mechanism of action: Binds to isoleucine (iles) trna synthetase MIC 8-256mg/L Mutation in iles gene High level resistance MIC 512mg/L Acquisition of mupa gene; novel isoleucine trna synthetase. Protein synthesis termination
No. items dispensed Resistance (%) Usage drives mutation Mupirocin usage and resistance in one Ward 120 100 80 60 40 20 80 70 60 50 40 30 20 10 0 0 2006-2006 - 2006-2006 - 2007-2007 - 2007-2007 - 2008-2008 - 2008-2008 - Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 p 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 R< = 18 S = 30
Macrolide resistance in Staphylococci /Streptococci Target modification erm gene methylates 23S Confers R to Macrolide-Lincosamidestype B Streptogramins (MLS B ) 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 Mechanism of action: Binds to 23S ribosomal subunit Protein synthesis termination
Macrolide resistance in Staphylococci /Streptococci - Detection ERYTH S CLINDA S Interpretation Type 1: Organism susceptible to both erythromycin and clindamycin Disc screen D-test R R Type 2: Organism resistant to erythromycin and clindamycin (constitutive MLSB) Placed 12-20mm apart Ery 15 ug Clin 2 ug R S Type 3: May have inducible resistance (inducible MLSB) or other mechanism 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 3 rd 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 Teicoplanin susceptibility R R R-r R r r r r r 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 Species ID important But not always vana genotype with vanb phenotype vand genotype with vanb phenotype E. gallinarum - vana / vanb E. raffinosus - vana
Susceptibility Testing 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. Glycopeptides MIC breakpoint (mg/l) S R > Disk content (µg) Zone diameter breakpoint (mm) S R < Teicoplanin 2 2 30 16 16 Vancomycin 4 4 5 12 12
Linezolid resistance in Enterococcus faecalis Resistance rare <1% Most commonly caused by mutation (G2576T) Resistance alert in June 2016 optra gene 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 Protein synthesis termination
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 Method dependant; perform microbroth dilution Borderline result of 2mg/L may have impaired clinical response Glycopeptides 1 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 Teicoplanin, Coagulase-negative staphylococci 2 4 2 4 Note A Note A 1. Glycopeptide MICs are method dependent and should be determined by Note A Note A 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 Telavancin, MRSA 0.125 2, 0.125 2, Note A Note A there may be an impaired clinical response. The resistant breakpoint has 3 3 been reduced to 2 mg/l to avoid reporting "GISA" isolates intermediate as Vancomycin, S. aureus Vancomycin, Coagulase-negative staphylococci 2 4 2 4 Note A Note A serious infections with "GISA" isolates are not treatable with increased Note A Note A 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.
Disc susceptibility testing GRSA Detected by disc
MIC Determination Microbroth dilution MIC <0.25 0.5 1 2 4 8 16 GSSA 10 5 inoculum Mueller Hinton Broth Incubation at 35±1 C in air for 18-20 hours Ab VAN TEIC MIC method hgisa GEOMean (mg/l) Range (mg/l) % resistance GSSA hgisa GISA GSSA hgisa GISA GSSA hgisa GISA GISA 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 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 Sn Sp
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 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 Detects all GISA, majority at 24h Cannot use to determine MIC Detects most hgisa and GISA 2% false positives
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 MHA MHA5T Method % GSSA correctly classified % hgisa correctly classified % GISA correctly classified MHA5T 97 78.7 93.8
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 10 6 / 10 3 10 6 / 10 3 10 6 / 10 3 10 3 / N 10 3 / N 10 3 / N
PAP-AUC Viable count Log cfu/ml PAP-AUC 2 Log viable count vs. VA conc n graph Ratio = AUC of test isolate/auc of Mu3 10 6 10 5 10 4 10 3 10 2 PAP of Mu 3 (hgisa), Mu50 (GISA), GSSA and Oxford Staphylococcus aureus 10 10 Mu 3 (hgisa) 10 9 Mu50 (GISA) 10 8 NCTC 6571 (GSSA) 10 7 GSSA 0.5 1 2 2.5 4 8 Vancomycin concentration mg/l PAP-AUC ratio criteria: 1.5 1.0 hvisa VISA VSSA GSSA < 0.9 hgisa 0.9-1.29 GISA 1.3 0.5 0.0 0 1 2 3 4 Observation number
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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