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Special Methods for

Detecting Antimicrobial
Resistance

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
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additional testing.

SCREENING TESTS (2) Special tests for detecting antibacterial resistance 4 .

SCREENING TESTS (3) Special tests for detecting antibacterial resistance 5 .

Enterococci .

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) 7 .

g. tobramycin. ≥2. e. and high level Gent and Streptomycin • Gentamicin .000 μg/ml for streptomycin and ≥ 500 μg. amikacin 8 • Streptomycin ..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.ml for gentamicin and is designated HLR (HLAR) • For routine AST: Pen and Vanco.

MICs. ≥ 16 μg/ml (VanA phenotype) • Moderate. MICs ≥ 64 μg/ml + Teicoplanin resistance. 32 µg/ml (R) Three most common phenotypes of Vancomycin resistance: • High-level. most commonly without teicoplanin resistance (VanB phenotype) 9 • Intrinsic low-level resistance associated with . MICs. 8 to 16 µg/ml (I).to high-level vancomycin resistance.Tests to Detect Resistance in Enterococci (3) Vancomycin Resistance CLSI MIC for Vanco: ≥4 µg/ml (S). ≥ 16 to 512 μg/ml .

E. casseliflavus. VanL genotypes have been found but are rare 10 . faecium. faecalis. gallinarum • VanD. E. VanG. VanE.Tests to Detect Resistance in Enterococci (4) • Both VanA and VanB phenotypes are most common in E. and E.

Tests to Detect Resistance in Enterococci (5) Screening methods for detecting vancomycin and high level aminoglycoside resistance in enterococci REFER TO CLSI M100-S25 TABLE 3F & 3I 11 .

Tests to Detect Resistance in Enterococci (6) VRE: Vancomycin agar screen test 12 .

Staphylococcus aureus .

aureusCLSI M100-S25 14 .Tests to Detect Resistance in S.

aureus (2) 15 .Tests to Detect Resistance in S.

aureus (3) 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 Page  16 .Tests to Detect Resistance in S.

aureus (4) • 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. 1 in 1M cells) 17 .Tests to Detect Resistance in S.g.

Tests to Detect Resistance in S. aureus (5) 18 .

other beta-lactam agents should be reported as (R) or should not be reported • mecA positive staphylococci are (R) to oxacillin (not cefoxitin). aureus isolated (MRSA • Oxacillin resistant staphylococci are resistant to all β-lactam agents.Tests to Detect Resistance in S. aureus (6) Report: Methicillin-resistant S. other β-lactams except those with anti-MRSA activity should be reported as (R) *Cefoxitin is used as a surrogate for mecAmediated oxacillin resistance 19 .

aureus ID Commercial Methods: Slide agglutination test for PBP2a (MRSA) 20 . aureus (7) Slide agglutination test for S.Tests to Detect Resistance in S.

Tests to Detect Resistance in S. aureus (8) MRSA: Oxacillin salt screen agar test 21 .

Tests to Detect Resistance in S. aureus (9) MRSA: Commercial Chromogenic agars 22 .

aureus (MRSA) isolated No Methicillin-resistant S. aureus (10) Reporting: Methicillin-resistant S. aureus isolated • MRSA +: Report Oxacillin as resistant • Notify clinician and infection control 23 .Tests to Detect Resistance in S.

aureus (11) Penicillin Resistance and β-Lactamase • Penicillin (R) strains of Staph produce βlactamase • 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 24 .Tests to Detect Resistance in S.

commercially available) • Perform these tests for isolate that test (S) to Penicillin using routine AST method • CLSI Interpretive criteria: ≥ 29 25 mm or .Tests to Detect Resistance in S. aureus (12) Tests for detection of β-lactamase production • Penicillin disk diffusion zone edge test • Nitrocefin-based test (disk.

aureus (13) Penicillin zone edge test More sensitive than Nitrocefin in detecting βlactamase production in S. aureus Sharp zone edge (cliff) = β-lactamase positive Resistant to penicillin. and ureidopenicillins REFER TO CLSI M100-S25 TABLE 3D Fuzzy zone edge (beach) = β-lactamase negative 26 .Tests to Detect Resistance in S. aminocarboxy-.

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) Commercial method. aureus (14) Nitrocefin β-lactamase test Chromogenic method Yellow (-) Red (+) Positive within 5 minutes to 1 hour For CoNS including S. follow manufacturer’s direction Report: β-lactamase positive Resistant to Penicillin… REFER TO CLSI M100-S25 TABLE 3D 27 .Tests to Detect Resistance in S.

aureus (15) 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 28 erm gene (usually ermA or ermC) .Tests to Detect Resistance in S.

aureus (16) Test for inducible clindamycin resistance (ICR) • In Staph. 18–24 h • ICR (+): Any growth in well 29 . Clinda (S) -> test for ICR • Broth microdilution method: 4 μg of erythromycin and 0. (MSLB) resistance is either constitutive or inducible • S. aureus: Erythromycin (R).5 μg of clindamycin/ml in same well 35°C.Tests to Detect Resistance in S.

Tests to Detect Resistance in S. aureus & S.Inducible lugdunensis D-Test Test for clindamycin • Use Erythromycin and Clindamycin disks 15-26 mm apart to detect inducible clindamycin resistance (ICR)resistance D-shape = Clinda Resistance 20 20 mm mm For beta-streptococci place disks 12-15 mm apart 12 mm E No D shape = Clinda susceptible CC . aureus (17) S.

” REFER TO CLSI M100-S25 TABLE 3G 31 . aureus (18) Reporting of ICR + strains • Report as “Clindamycin resistant” • Include comment: “This isolate is presumed to be resistant based on detection of inducible clindamycin resistance.Tests to Detect Resistance in S.

024 μg/ml • Vancomycin-intermediate S. ≥16 μg/ml (R) • Interpretive categories for CoNS including S. aureus: ≤2 μg/ml (S). lugdunensis: ≤ 4 μg/ml (S) 8 to 16 μg/ml (I).Tests to Detect Resistance in S. 4 to 8 μg/ml (I). aureus – VISA due to changes in the cell wall. 32 no van . not the van gene • CoNS (VISS) – due to cell wall changes. aureus – VRSA MICs range from 32 to 1. and ≥32 μg/ml (R) • Vancomycin resistant S. aureus (19) Vancomycin Resistance • CLSI vancomycin interpretive categories for S.

Coagulase-negative Staphylococcus .

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) 34 .Tests to Detect Resistance in CoNS Fox disk: ≤ 24mm = mecA + ≥ 25mm = mecA - Broth microdilution: >4 μg/ml = mecA + ≤ 4 μg/ml= mecA - Β-lactamase test: If test is negative.

Streptococcus pneumoniae .

I or S Always confirm with MIC . pneumo from CSF Oxacillin 19 mm = Pen R.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.

lactamase test 37 .Other Organisms Β.

Thank you 38 .