DETECTION OF RESISTANCE IN

GRAM POSITIVE ORGANISMS

Dr.S.T.Renuka

postgraduate
 CLSI guidelines for Staphylococcus spp
Tier 1 Tier 2 Tier 3 Tier 4
Azithromycin or
Clarithromycin or Penicillin Ceftaroline Cipro/levo/
Erythromycin Daptomycin Tedizolid moxifloxacin
Linezolid Rifampin Dalbavancin
Clindamycin
Lefamulin Oritavancin
Oxacillin Telavancin
Cefoxitin(surrogate) Gentamicin
Marker for oxacillin)

Doxy/Mino/
Tetracycline

Trimethoprim-
sulfamethoxazole
Vancomycin

Urine only-
Nitrofurantoin
FOOT NOTES
 Macrolides,clindamycin,Minocycline – not reported for urinary
specimens

 Organism susceptible for Tetracycline are also S to

Doxy & minocycline ,If I or R may be S to Doxy or minocycline or both.

 Only MIC – Vancomycin,Rifampin,Dalbavancin,Oritavancin,

Telavancin.

 Daptomycin – not for respiratory specimens

TYPES OF RESISTANCE
Staphylococcus spp
• Beta lactamase
• BORSA
• MRSA
• GISA/VISA
• VRSA
• Inducible Clindamycin resistance
• High level Mupirocin resistance

Enterococcus spp
• High level amino glycoside resistance
• VRE
 Test for detection of
Betalactamase in
Staphylococcus
 spp
Disk diffusion (Penicillin zone edge test)

S. aureus with MHA

MIC <0.12 16-18 hrs of
µgm/ml or Zones 10 Units disk
Incubation
>29mm

Beta Beta
lactamas lactamas
e e
Positive- Negative
Sharp – Fuzzy
zone zone
edge(clif edge(bea
f) ch)
A – Beta lactamase positive
B- Beta lactamase negative
Nitrocefin based test

S. aureus with MIC <0.12 µgm/ml

or Zones >29mm

Growth taken from zone margin

around a penicillin disk

Conversion from yellow to

pink/red – positive
Nitrocefin test
Qc:
positive control :S.aureus ATCC 29213
Negative control : S.aureus ATCC 25923
Reporting:
Nitrocefin positive – Report as Positive for Beta lactamase
Nitrocefin negative – Perform Zone edge test and report
Penicillin susceptibility especially in Endocarditis isolates.
Beta lactamase positive staphylococci are resistant to
penicillin, amino- ,carboxy- penicillins and
ureidopenicillins.
BORSA
 Borderline Oxacillin Resistance to S.aureus –
Oxacillin resistant but cefoxitin susceptible
and negative for Mec A/C gene.
 Usually susceptible to other antimicrobial
drugs
 Due to increased production of beta
lactamase mediated by non mec A gene .
MRSA
 It is mediated by chromosomaly coded gene called
mecA gene, which alters PBP to PBP2a present in large
mobile staphylococcal elements called SCC mecA.
 Altered PBP2a has less affinity for Betalactam
antibiotics – Resistant to all
 Mec C gene coding for PBP2c also a/w MRSA
 Strains with mecC are typically cefoxitin resistant and
oxacillin susceptible.
 MecC resistance cannot be detected by tests directed
at mecA or PBP2a.
Community Hospital associated
associated MRSA MRSA
 Mec A gene subtype  Mec A gene subtype
IV,V,VI I,II,III
 They are multi drug
 Usually more
resistant(less virulent)
virulent and
 Cause perioperative
express Panton
wound infections and
valentine toxin
nosocomial outbreak.
 Cause invasive skin  Hospital staff are
infection such as major carriers
necrotizing fasciitis
Detection of MRSA

CLSI RECOMMENDED:
1. Disk diffusion: Oxacillin and Cefoxitin diffusions test
2. Oxacillin Salt Agar Screening method
3. Dilution method: Agar dilution and Broth dilution
OTHERS:
4. Chromagar
5. E test method
6. Latex agglutination – detect PBP2a
7. Automated methods (VITEK2 system- Cefoxitin screening)
8. Molecular methods – detection of mecA gene
 Cefoxitin disk diffusion
method
SPECIES SUSCEPTIBLE INTERMEDIATE RESISTANT

S.aureus ≥22mm - ≤21mm

S.lugdenensis ≥22mm - ≤21mm

Other CoNS ≥25mm - ≤24mm

Cefoxitin Disk- 30µg procedure read after 24 hrs

(18 hrs if R)
Isolates that are positive for mecA mediated resistance
should be reported as methicillin(not cefoxitin)
resistant and other beta lactam agents except
ceftaroline should be reported as resistant or should
not be reported
Oxacillin disk diffusion method
 Only indicated in species for S.epidermidis, S.psuedintermedius,
S.schleiferi
 1µgm oxacillin disk on MHA for 16 to 18 hrs

≤17mm-positive
for mecA
mediated ≥18mm- negative for
resistance mecA mediated
resistance
Oxacillin salt agar screening
for S.aureus only
 MHA with 4% Nacl ; 6µgm/ml oxacillin
 Colony suspension to 0.5 MC farland turbidity. Add 1 µg/ml and spot an
area 10-15 mm diameter.
 After 24 hrs of incubation with transmitted light
 > 1 colony - positive for mec A mediated resistance
Broth Microdilution using
cefoxitin
 Cation adjusted MHB with 4µgm/ml cefoxitin
 Standard broth microdilution procedure
 Read after 16- 20 hrs of incubation.

 ≥ 8µgm/ml - positive for mec A mediated resistance

 ≤4µgm/ml - negative for mecA mediated resistance
Broth Microdilution and agar
dilution using oxacillin
For S.aureus and S.lugdunensis other spp

CAMHB with 2% Nacl CAMHB with 2% Nacl

MHA with 2% Nacl(agar dilution) MHA with 2% Nacl(agar dilution)

2 µgm/ml oxacillin 0.5 µgm/ml oxacillin

Standard procedure after 24 hrs (18 Standard procedure after 24 hrs (18 hrs
hrs if R) if R)

≥4µgm/ml - positive for mec A ≥ 1 µgm/ml - positive for mec A

mediated resistance mediated resistance

≤2µgm/ml - negative for mecA ≤0.5 µgm/ml - negative for mecA

mediated resistance mediated resistance
ORGANISM Cefoxiti Cefoxitin Oxacillin Oxacillin Oxacillin
n MIC Disk MIC Disk Salt agar
diffusion diffusion

S.aureus Yes (16- Yes (16- Yes (24hrs) No Yes (24hrs)

20hrs) 18hrs)

S.lugdenensis Yes (16- Yes (16- Yes (24hrs) No No

20hrs) 18hrs)
S.epidermidis No Yes (24hrs) Yes (24hrs) Yes (16- No
18hrs)

S.pseudinter No No Yes (24hrs) Yes (16- No

medius 18hrs)
S.schleiferi No No Yes (24hrs) Yes (16- No
18hrs)
Other staph No Yes (24hrs) Yes (24hrs) No No
spp not listed
above
Chromagar
 It contains chromogenic substartes and various
inhibitors that combine primary growth and selective
detection of MRSA

 EX: MRSA ID, MRSA Select, CHROMagar MRSA ,Denim

Blue.

 Not all strain will grow and can enhanced by placing

cefoxitin disk by detecting growth around the disk.
 VITEK Testing of S.aureus

Oxacillin MIC Cefoxitin MIC Confirmation Interpretati

on

S S No need MSSA(MecA -
VE)

R R No need MRSA(MecA
+VE)

S R Cx DD MRSA(MecC
+VE)

R S Ox screen agar BORSA

 Vancomycin

 MIC should be done to test Vancomycin in all

Staphylococcal species.
 Disk method cannot differntiate VA- I & VA- R
and zone diameters do not correlate with VA MIC.

 It will detect VRSA containing vanA determinant

alone.
 Does not differentiate wild type vs non VanA
resistance.
MIC of Vancomycin
Susceptible intermediate Resistant

Vancomycin S.aureus ≤2µg/ml 4-8µg/ml ≥16µg/ml

Including
MRSA

Other ≤4µg/ml 8-16µg/ml ≥32µg/ml

staphylococc
al spp

VISA – 4-8µg/ml
Vancomycin screen agar

 Agar dilution – BHI agar: VA 6µg/ml

 Examine after 24 hrs of incubation.
 >1 colony – presumptive reduced susceptibility to
vancomycin
 Testing on BHI VA screening agar doesn’t reliably detect
all VISA . Some strains for which VA MIC are 4 µg/ml will
fail to grow.
 Perform Vancomycin MIC to determine MIC of S.aureus
that grow on BHI-Vancomycin screen agar.
Inducible Clindamycin
resistance
 It can occur through efflux or methylation of their
ribosomal target site typically mediated by
erm(erythromycin ribosome methylase) genes.
 Two types: constitutive, Inducible
 Constitutive- rRNA methylase is always produced, in
S.aureus with consititutive resistance, will be resistant
to both
 Inducible- methylase is produced only in the presence of
inducing agent (Erythro is an effective inducer of MLSB
resistance).so invitro R-E & S- Cd
Mechanism Resistance Erythromycin Clindamycin
gene

Efflux msrA R S
D zone test
(-)

Ribosome ermA or ermC R S R

alteration (inducible)
D zone test
(+)

Ribosome ermA or ermC R R (constitutive)

alteration D zone test
(-)
 Inducible clindamycin
resistance- Disk diffusion method

All Staphylococcus spp S.Pneumoniae and other Beta –

hemolytic streptococcus spp.

MHA/blood agar MHA with 5% blood or TSA

15µg erythromycin & 2µg clindamycin 15µg erythromycin & 2µg clindamycin
disk 15-26mm apart after 16- 18 hrs disk 12mm apart after 20-24 hrs

Flattening of zone of inhibition adjacent to erythromycin disk (D zone ) = ICR

Hazy growth with in zone of inhibition around clindamycin = Clindamycin
resistance even if no D zone is apparent.

Reporting: “This isolate is presumed to be resistant based on detection of

ICR as determined by testing Clindamycin in combination with
erythromycin”
Inducible clindamycin resistance-
Broth dilution method

All Staphylococcus spp S.Pneumoniae and other Beta –

hemoltic streptococcus spp.

CAMHB CAMHB with lysed horse blood

4 µg/ml erythromycin & 0.5 µg/ml 1µg/ml erythromycin & 0.5 µg/ml
clindamycin in same well clindamycin in same well

Any growth = ICR No growth = no ICR

Test for Detecting High level
mupirocin resistance
Disk Diffusion Broth microdilution

MHA CAMHB

200µg Mupirocin disk 256 µg/ml

Std procedure and read after 24 hrs Std procedure and read after 24 hrs

Examine growth within zone of For single 256 µg/ml well

inhibition.
No zone – high level mupirocin Growth – High level mupirocin
resistance resistance
Any zone – absence of high level No growth - absence of high level
resistance resistance
Enterococcus
 CLSI guidelines for Enterococcus spp
Tier 1 Tier 2 Tier 3 Tier 4
Ampicillin Vancomycin Tedizolid
Penicillin Dalbavancin
Gentamycin Streptomycin Oritavancin
(High level (High level Telavancin
Urine only- resistance testing resistance testing
Nitrofurantoin only) only)

Daptomycin Urine only-

Linezolid Fosfomycin
Tetracycline
Urine only-
Ciprofloxacin
Norfloxacin
Footnotes
 Ampicillin S S to Amoxicillin,Amoxy – clav,ampi-sulbactam,piptaz
among non beta lactamase producing enterococci.
 Similarly penicillin S S to ampicillin, Amoxicillin,Amoxy – clav,ampi-
sulbactam,piptaz among non beta lactamase producing enterococci.
 But if ampicillin S cant be assumed to be Penicillin S.

Aminoglycosides(except high level testing), Cephalosporins,

clindamycin,Cotrimoxazole may appear active invitro, but not
effective clinically, and isolates should not be reported as
Susceptible.
High level Aminoglycoside
resistance - Gentamycin
Disk diffusion Broth micro Agar dilution
dilution
MHA BHI broth BHI agar

120µg gentamycin 500µg/ml 500µg/ml

disk gentamycin gentamycin

Std disk diffusion Std micro dilution 10µl of a 0.5 mc

procedure procedure Farland
suspension spotted
onto agar surface
16-18 hrs 24 hrs 24hrs

6mm= resistant Any growth = >1 colony=

7-9mm= resistant resistant
inconclusive
>= 10 mm -
susceptible
High level Aminoglycoside
resistance - streptomycin
Disk diffusion Broth micro Agar dilution
dilution
MHA BHI broth BHI agar

300 µg 1000µg/ml 2000µg/ml

streptomycin disk Streptomycin Streptomycin
Std disk diffusion Std micro dilution 10µl of a 0.5 mc
procedure procedure Farland suspension
spotted onto agar
surface
16-18 hrs 24 – 48 hrs 24 – 48 hrs

6mm= resistant Any growth = >1 colony= resistant

7-9mm= resistant
inconclusive
>= 10 mm -
susceptible
Reporting
• It is not synergistic with cell wall active agent
R (ampicillin,penicillin,vancomycin)

• It is synergestic with cell wall active agent

S (ampicillin,penicillin,vancomycin) that is also susceptible

• If disk diffusion is inconclusive perform an agar dilution

or broth dilution MIC test to confirm

Other aminoglycosides need not to be tested because their activities

against enterococci are not superior to gentamycin and streptomycin
Vancomycin resistant
enterococci
 Vancomycin susceptible – D-alanyl-D –alanine
 Glycopeptide resistant – D-alanyl-D – lactate or
D-alanyl-D –serine.

Gene clusters code • vanA,

for vanB,vanD,vanM
D-alanyl-D – lactate

Gene clusters code • vanC1,vanC2,vanC3,

for D-alanyl-D – vanE,vanG,vanL,vanN
serine
 Van A – Vancomycin and teicoplanin
inducible , high resistance to both
 Van B- Vancomycin inducible resistance to
various concentration but susceptible to
teicoplanin.
 VanC – intrinsic, constitutive resistance to
vancomycin and susceptible to teicoplanin.
This gene responsible for intrinsic
resistance to glycopeptide resistance in
E.gallinarum,E.casseliflavus,E.flavescens .
 Disk diffusion method MIC break points
vancomy S I R S I R
cin
30µg ≥17 15- ≤14mm ≤4 8-16 ≥32
mm 16mm µg/ml µg/ml µg/ml

For testing VRE ,plates should be incubated

for full 24hrs .Presence of any haze or any
growth with in zone of inhibition is
resistance.
Vancomycin screen agar
 Agar dilution – BHI agar: VA 6µg/ml
 Examine after 24 hrs
 >1 colony – presumptive vancomycin
resistance.
 Perform MIC for colonies grown on BHI
agar and test for motility and pigment
production to distinguish acquired
resistance(vanA,vanB) from intrinsic
intermediate resistance to vancomycin
(vanC) ex:
E.gallinarum,E.casseliflavus,E.flavescens
Streptococcus pneumoniae
Footnotes

 If S to Erythro then S to azithro and clarithromycin

 Not for urinary isolates – Macrolides and clindamycin
 CSF isolates – MIC of
Penicillin,Cefotaxime,ceftriaxone,Meropenam should be
done routinely and Vancomycin either by MIC or DD.
 Only MIC – Cefipime,Ertapenem,Imipenam
 S to Levofloxacin then S to Gemi,moxifloxacin.
Footnotes
 AST for Beta haemolytic streptococci and S.pyogenes
not routinely done as non susceptible isolates are
extremely rare and if found resistant should retest and
if confirmed submitted to Public Health Laboratory.
 Intrapartum prophylaxis for Grp B streptococci:
Penicillin or ampicillin.
 Low risk for penicillin allergy – Cefazolin
 High risk for penicillin allergy – Clindamycin
 If isolates from high risk for penicillin allergy ICR test
should be performed but ONLY CLINDAMYCIN should be
reported , Erythromycin for testing alone.
CLOVIBACTIN – from Bacterial dark
matter

 Using the device, called iCHip, the US

researchers discovered Clovibactin in a
bacterium isolated from a sandy soil from North
Carolina: Eleftheria terrae ssp. Carolina.
 NovoBiotic Pharmaceuticals shows that
Clovibactin successfully treated mice infected
with the superbug Staphylococcus aureus.
 The multi-target attack mechanism of
Clovibactin blocks bacterial cell wall synthesis
simultaneously at different positions. This
improves the drug's activity and substantially
increases its robustness to resistance
development.
 Upon binding the target molecules, it
self-assembles into large fibrils on the
surface of bacterial membranes.

 These fibrils are stable for a long time

and thereby ensure that the target
molecules remain sequestered for as long
as necessary to kill bacteria.
 These fibrils only form on bacterial
membranes and not toxic to human cells.
THANK YOU