SPECIAL PHENOTYPIC METHODS FOR DETECTING

ANTIMICROBIAL RESISTANCE
By: Joselle N.Ealdama , RMT

PURPOSE:
 To monitor organisms with unusual susceptibility patterns by recognition of different
resistance mechanisms present in the bacteria through special phenotypic methods.
 Used for detecting antibacterial resistance ranges from the rapid and simple spot ß-
lactamases test to the more time-consuming and complex test either:
1. Supplemental or traditional testing depending on the organism and assay
used.
2. Disk approximation tests incorporated during routine susceptibility testing.

Importance of Performing Phenotypic Methods

 Characterize an isolate as susceptible or resistant to one or more antimicrobial agents
based on a specific resistance mechanism or phenotype.
 Some tests have sufficient sensitivity & specificity such that results of the screen test can
be reported without additional testing.
 Other tests require confirmatory testing as to presumptive results obtained.

Special Phenotypic Methods

These tests are include the following:
1. Detection of Extended Spectrum ßeta-lactamase (ESBL) among E.coli, Klebsiella spp,
and Proteus spp.
2. Detection of ampC beta-lactamase among Enterobacter, Serratia, Citrobacter, Hafnia,
Aeromonas, Morganella, Providencia (ESCHAMP)
3. Detection of Metallo-beta-lactamase (MBL) or Carbapenemase in Enterobacteriaceae;
P. aeruginosa and other Non-fermenting Organisms
4. Detection of Penicillin Resistance in Streptococus pneumoniae using Oxacillin disk 1 ug
5. Detection of mecA-mediated Oxacillin Resistance in Staphylococci
6. Detection of Inducible Clindamycin Resistance (ICR) in Staphylococci

Detection of Extended Spectrum Beta-lactamase (ESBL) among E.coli, Klebsiella

spp, and Proteus spp.

ESBL DETECTION
- ESBL’s are enzymes that mediate resistance to extended –spectrum cephalosporins
and monobactams but do not affect cephamycins or carbapenems.
- Presence of an ESBL producing organism in a clinical infection can result in treatment
failure if one of the above classes of drugs is used.
- ESBL production can be detected in the lab by disk potentiation test.
o Placing a ATM 30ug disk and AMC 20/10ug 1.5cm apart.
o An elliptical clearing or keyhole effect between to disks indicates the inhibition of
ß-lactamase or ESBL production.
- This is present and detected among Enterobacteriaceae
 ESBL Detection using
CT/CTL
ESBL (+) = CT/CTL value of > 8ug/ml
ESBL (-) = CT/CTL value of < 8ug/ml

Detection of ampC beta-lactamase among Enterobacter, Serratia, Citrobacter,

Hafnia, Aeromonas, Morganella, Providencia (ESCHAMP)

ampC DETECTION
- ampC beta-lactamase can be detected by observing the phenotypic expression such as
flattening of zone of inhibition of cefotaxime 30ug disk against imipenem 10ug disk
- Confer resistance to beta-lactam inhibitor (no synergy)
- Confer resistance to cefoxitin (FOX)
- Not inducible by beta-lactams
Detection of Metallo-beta-lactamase (MBL) or Carbapenemase in
Enterobacteriaceae; P. aeruginosa and other Non-fermenting Organisms

1. Modified Hodge Test (MHT)

Purpose:
- This test can detect carbapenemase among Enterobacteriaceae

Principle:
- Carbapenemase production is detected by the MHT when the test isolate produces the
enzyme and allows growth of a carbapenem susceptible strain(ECO ATCC 25922)
towards a carbapenem (ertapenem 10ug)disk.
- The result is a characteristic “clover-leaf” like indentation of E.coli growing along the test
organism growth streak within the disk diffusion zone.

2. Imipenem-EDTA double disk synergy test

Purpose:
- This test can detect metallo-β-lactamases among P.aeruginosa, Acinetobacter species,
and other
Principle: non-Enterobacteriaceae.
- Carbapenems like imipenem, meropenem and ertapenem contains enzymes which are
inhibited by a chelating agent (EDTA). This interaction aids in the detection of
carbapenemases which are zinc-dependent metallo-enzymes that hydrolze the
penicillins.
MBL Detection using Imipenem-EDTA disks MBL Detection using Imipenem-
Imipenem/Imipenem+EDTA Etest

Interpretation of Results:
MBL (+)=Keyhole between imipenem MBL (+) = IP/IPI value of > 8 ug/ml
and imipenem-EDTA disk
MBL (-)=No keyhole between imipenem MBL (-) = IP/IPI value of <8 ug/ml
and imipenem-EDTA disk

Detection of Penicillin Resistance in Streptococcus pneumoniae using Oxacillin

disk 1ug

Oxacillin 1ug Disk Screen

Purpose:
- This disk is used to screen for penicillin resistance among S. pneumoniae.
- If test Result for oxacillin 1 ug is ≤19 mm, perform confirmatory test.

Confirmatory Test:
Penicillin G, Cefotaxime and Ceftriaxone MIC.
Oxacillin 1ug Disk Screen

Penicillin Resistant (Oxacillin) = Oxacillin zone diameter of < 19mm

Penicillin Susceptible (Oxacillin) = Oxacillin zone diameter of >20mm

Detection of mecA-mediated Oxacillin Resistance in Staphylococci

Detection mecA-mediated Oxacillin

- mecA or the penicillin-binding protein 2a also calledPBP2a are the most accurate
methods for prediction of resistance to oxacillin
- Staphylococci that carry the mecA gene, or produce PBP2a should be reported as
oxacillin resistant.
- Can be detected by testing cefoxitin 30ug and is used as surrogate for mecA-mediated
oxacillin.

Detection mecA-mediated Oxacillin

mecA positive = S. aureus and S. lugdunensis: cefoxitin disk diffusion zones are <21

mecA negative = S. aureus and S. lugdunensis: cefoxitin disk diffusion zones are >22

- Isolates that test as mecA positive should be reported as oxacillin (not cefoxitin)
resistant; other beta-lactam agents should be reported as resistant or should not be
reported.

Resistance mechanisms that contribute to oxacillin resistance among Staphylococcus

species
- production of supplemented PBP (PBP2a) encoded by a chromosomal mecA gene,
- inactivation of the drug by increased production of ß-lactamase,
- production of modified intrinsic PBP’s with low affinity for the drug.

Detection of Inducible Clindamycin Resistance (ICR) in Staphylococci

Detection of Inducible Clindamycin Resistance

- Macrolide resistant isolates of S. aureus and coagulase-negative Staphylococcus

species may have an inducible resistance to clindamycin [methylation of 23S rRNA
encoded by the erm gene also referred to as macrolide, lincosamide and type B
streptogramin resistance (MLSB)] or may be resistant only to macrolides.