Lecture #6. Special ASTs_Indonesia 2015

Special Methods for Detecting Antimicrobial Resistance

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OUTLINE

Special tests to detect resistance in• Enterococci• Staphylococci• Pneumococci• Enterobacteriaceae*• Other organisms

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SCREENING TESTS

• Screening tests detects specific resistance mechanism or phenotype in certain bacterial strains

• characterize an organisms susceptibility or resistance to an antimicrobial agent

• These tests do not provide an MIC of the antimicrobial agent

• some have sufficient sensitivity and specificity that confirmation of the result is unnecessary

• some results can be reported without additional testing.

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SCREENING TESTS (2)Special tests for detecting antibacterial resistance

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SCREENING TESTS (3)Special tests for detecting antibacterial resistance

Enterococci

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Tests to detect resistance in Enterococci

HLAR• Pen/Vanco + Gent/Streptomycin for treatment of

serious invasive enterococcal infections• These agents act synergistically to enhance killing• Aminoglycosides cannot be used as single agents for

therapy due to poor activity against enterococci• Intrinsic, moderate level resistance (MICs range from

8 to 256 mg/ml)

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Tests to detect resistance in Enterococci (2)

HLAR• Aminoglycoside resistance usually corresponds to

MIC’s that are significantly above the concentration normally tested in routine susceptibility tests, e.g., ≥2,000 μg/ml for streptomycin and ≥ 500 μg.ml for gentamicin and is designated HLR (HLAR)

• For routine AST: Pen and Vanco, and high level Gent and Streptomycin

• Gentamicin , tobramycin, amikacin• Streptomycin

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Vancomycin ResistanceCLSI MIC for Vanco: ≥4 µg/ml (S), 8 to 16 µg/ml (I), 32 µg/ml (R)Three most common phenotypes of Vancomycin resistance:• High-level, MICs ≥ 64 μg/ml + Teicoplanin resistance,

MICs, ≥ 16 μg/ml (VanA phenotype)• Moderate- to high-level vancomycin resistance, MICs, ≥

16 to 512 μg/ml , most commonly without teicoplanin resistance (VanB phenotype)

• Intrinsic low-level resistance associated with E. gallinarum and E. casseliflavus , MICs 2 to 32 mg/ml (VanC phenotype)

Tests to Detect Resistance in Enterococci (3)

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• Both VanA and VanB phenotypes are most common in E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum

• VanD, VanE, VanG, VanL genotypes have been found but are rare


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Screening methods for detecting vancomycin and high level aminoglycoside resistance in enterococci

REFER TO CLSI M100-S25 TABLE 3F & 3I


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VRE: Vancomycin agar screen test


Staphylococcus aureus

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Tests to Detect Resistance in S. aureus CLSI M100-S25

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Tests to Detect Resistance in S. aureus (2)

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Oxacillin Resistance & mecA-mediated Ox resistance:• MRSA still commonly used even though methicillin is

no longer available for treatment• Most resistance to Oxacillin in Staphylococci are

mediated by the mecA gene• mecA directs the production of a supplemental

penicillin binding protein (PBP2a) during bacterial cell replication


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• Resistance is expressed either homogeneously or heterogeneously

• Homogeneous – nearly all bacterial cell progeny express the resistance phenotype – easier to detect with standard AST methods

• Heterogenous – only a fraction of the progeny population express resistance (e.g. 1 in 1M cells)


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Report: Methicillin-resistant S. aureus isolated (MRSA• Oxacillin resistant staphylococci are resistant to all β-

lactam agents; other beta-lactam agents should be reported as (R) or should not be reported

• mecA positive staphylococci are (R) to oxacillin (not cefoxitin); other β-lactams except those with anti-MRSA activity should be reported as (R)

*Cefoxitin is used as a surrogate for mecA-mediated oxacillin resistance


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Commercial Methods: Slide agglutination test for PBP2a (MRSA)

Slide agglutination test for S. aureus ID

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MRSA: Oxacillin salt screen agar test


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MRSA: Commercial Chromogenic agars


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Reporting:Methicillin-resistant S. aureus (MRSA) isolatedNo Methicillin-resistant S. aureus isolated

• MRSA +: Report Oxacillin as resistant• Notify clinician and infection control


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Penicillin Resistance and β-Lactamase • Penicillin (R) strains of Staph produce β-lactamase• Some β-lactamase producing staphylococcal isolates

test susceptible to penicillin (MIC ≤ 0.12 µg/ml or zone diameters ≥ 29mm)

• Staphylococcal β-lactamase is readily inducible


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Tests for detection of β-lactamase production• Penicillin disk diffusion zone edge test• Nitrocefin-based test (disk, commercially

available)• Perform these tests for isolate that test (S) to

Penicillin using routine AST method• CLSI Interpretive criteria: ≥ 29 mm or ≤ 0.12

µg/ml (S); ≤ 28mm or ≥ 0.25 µg/ml (R)


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More sensitive than Nitrocefin in detecting β-lactamase production in S. aureus

Penicillin zone edge test

Sharp zone edge (cliff) =β-lactamase positiveResistant to penicillin, amino- carboxy-, and ureidopenicillins

Fuzzy zone edge (beach) = β-lactamase negative


REFER TO CLSI M100-S25 TABLE 3D

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Commercial method, follow manufacturer’s direction

Nitrocefin β-lactamase testChromogenic method

Yellow (-) Red (+)

Positive within 5 minutes to 1 hour

For CoNS including S. lugdunensis: use induced growth as inoculum for testing(growth taken from the zone margin surrounding a penicillin or cefoxitin disk test on MHA plate after 16-18 hours incubation)


REFER TO CLSI M100-S25 TABLE 3D

Report: β-lactamase positive Resistant to Penicillin…

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Test for inducible clindamycin resistance (ICR)• Clindamycin (lincosamide); Erythromycin (macrolide) • Mechanisms of action and resistance for

Erythromycin and Clindamycin are similar• M type mechanism confers resistance to macrolide

only• MSLB type mechanism confers resistance to

macrolides, lincosamide and streptogramin B agents mediated by an erm gene (usually ermA or ermC)


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Test for inducible clindamycin resistance (ICR)• In Staph, (MSLB) resistance is either constitutive or

inducible• S. aureus: Erythromycin (R), Clinda (S) -> test for ICR • Broth microdilution method: 4 μg of erythromycin

and 0.5 μg of clindamycin/ml in same well 35°C; 18–24 h• ICR (+): Any growth in well


S. aureus & S. lugdunensis D-Test• Use Erythromycin and Clindamycin disks 15-26 mm apart to detect inducible clindamycin resistance

D-shape = Clinda Resistance

No D shape = Clinda susceptible

20 mm

12 mmE CC

For beta-streptococci place disks 12-15 mm apart

Tests to Detect Resistance in S. aureus (17)Test for Inducible clindamycin resistance (ICR)

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Reporting of ICR + strains• Report as “Clindamycin resistant”• Include comment:

“This isolate is presumed to be resistant based on detection of inducible clindamycin resistance.”

REFER TO CLSI M100-S25 TABLE 3G

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Vancomycin Resistance • CLSI vancomycin interpretive categories for S. aureus:

≤2 μg/ml (S), 4 to 8 μg/ml (I), ≥16 μg/ml (R) • Interpretive categories for CoNS including S. lugdunensis:

≤ 4 μg/ml (S) 8 to 16 μg/ml (I); and ≥32 μg/ml (R) • Vancomycin resistant S. aureus – VRSA MICs range from 32 to 1,024 μg/ml• Vancomycin-intermediate S. aureus – VISA

due to changes in the cell wall, not the van gene• CoNS (VISS) – due to cell wall changes; no van gene-mediated

vancomycin resistance reported

REFER TO CLSI M100-S25 TABLE 3F


Coagulase-negative Staphylococcus

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Tests to Detect Resistance in CoNS

Β-lactamase test:If test is negative, use induced growth as inoculum for testing(growth taken from the zone margin surrounding a penicillin or cefoxitin disk test on MHA plate after 16-18 hours incubation)

Fox disk: Broth microdilution:≤ 24mm = mecA + >4 μg/ml = mecA +≥ 25mm = mecA - ≤ 4 μg/ml= mecA -

Streptococcus pneumoniae

Detection of Penicillin Resistance in Streptococcus pneumoniae

• Use Mueller-Hinton + 5% sheep blood agar• Incubate in CO2 instead of air• Report actual MIC

Report both Pen & Cefotaxime or Ceftriaxone & Mero MICs for S. pneumo from CSF

Oxacillin 19 mm = Pen R, I or S

Always confirm with MIC

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Other Organisms

Β- lactamase test

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Thank you

Documents

Lecture #6. Special ASTs_Indonesia 2015