ANTIBIOTIC

RESISTANCE IN GPC - II
DR APARNA GOPINATH
CONTENTS
• VRSA ,VISA,hVISA
• Vancomycin Resistance Detection
• hVISA : Detection methods
• High Level Mupirocin resistance

• Vancomycin Resistance detection in Enterococci

• High level Aminoglycoside resistance screening

• Resistance detection in Pneumococci

• Vancomycin-resistant Staphylococcus
aureus (VRSA) are strains
of Staphylococcus aureus that have
acquired resistance to the
glycopeptide antibiotic vancomycin.

• Resistance in VRSA is conferred by the

plasmid mediated vanA gene and operon

• Acquired resistance through uptake of a

vancomycin resistance gene cluster
from Enterococcus (VRE)
Two key events are necessary
for vanA operon-mediated
vancomycin resistance:

1) hydrolysis of dipeptide D-Ala-D-

Ala peptidoglycan precursors,
which bind vancomycin, and
2) synthesis of D-Ala-D-lactate
peptidoglycan precursors, which
cannot bind vancomycin
Vancomycin MIC in Staphylococcus aureus
≤ 2 µg/mL. Susceptible

4 to 8 µg/mL Intermediate

≥ 16 µg/mL Resistant
• Vancomycin-intermediate S. aureus (VISA)
GISA (glycopeptide-intermediate Staphylococcus aureus),
indicating resistance to all glycopeptide antibiotics.

These bacterial strains presents as thickening of the cell wall, which is

believed to reduce the ability of Vancomycin to diffuse into the cell
Heterogeneous Vancomycin-
intermediate S. aureus (hVISA)

• An hVISA phenotype refers to a mixed cell

population—derived originally from a single
colony of S. aureus—in which the majority of
cells have little or no resistance to vancomycin
(MIC ≤ 2 µg/ml) and a sub-population of cells is
resistant to the antibiotic at the level of VISA (MIC
≥ 4 µg/ml)
VRSA • MIC

VISA • MIC

• MET
hVISA • PAP AUC METHOD
 Vancomycin Broth
dilution

Vancomycin Agar
Vancomycin dilution
MIC
Resistance

Vancomycin
Screen Agar
• Optimum MIC range for testing Vancomycin should be 0.25μg/mL to
64 μg/mL
 Broth Dilution Agar Dilution

Media CAMHB MHA

CAMHB+2%NaCl MHA+2%NaCl
CAMHB +Ca

Inoculum Colony suspension to obtain Colony suspension to obtain

0.5 McFarland turbidity 0.5 McFarland turbidity

Incubation 35°C±2°C; ambient air x 24h 35°C±2°C; ambient air x 24h

 Vancomycin Screen Agar
Category Details
Test Method Agar dilution
Organism Group S. aureus
Medium BHI agar
Antimicrobial Concentration 6 μg/mL vancomycin
Inoculum Colony suspension to obtain 0.5 McFarland turbidity
Incubation Conditions 35°C±2°C; ambient air
Incubation Length 24 hours

Examine carefully with transmitted light for > 1 colony or light film of growth. > 1
Results
colony = presumptive reduced susceptibility to vancomycin

Perform a vancomycin MIC using a validated MIC method to determine

Additional Testing and Reporting
vancomycin MICs on S. aureus that grow on BHI-vancomycin screening agar.

Quality Control Strains E. faecalis ATCC® 29212 - susceptible, E. faecalis ATCC® 51299 - resistant
Limitations of the procedure
• Testing on BHI-vancomycin screen agar does not reliably detect all
Vancomycin intermediate S. aureus strains.

• Some strains for which the vancomycin MICs are 4μg/ml will fail to
grow.
Vancomycin MIC MIC Method Disk Diffusion method Vancomycin Screen
Agar
<=2 YES NO YES

4 YES NO VARIABLE

8 YES NO YES

16 YES NO YES

32 YES YES YES

hVISA
• Indication for Screening of hVISA : Bacteremia/ deep seated
infections,failing antibiotic therapy, persistent positive cultures on
therapy.
Colony morphology
• <1 in 105 – 106 bacteria
• Reduced pigmentation
• Smaller colony size
• Slow growing

• ROUTINE MIC CANNOT BE DONE

 Detection of hVISA

Screening test Confirmatory methods

Population Analysis
Macromethod E Test Screening Agar Profile Assay (PAP Modified PAP Method
(MET) Assay)

E TEST
GLYCOPEPTIDE
MHA5T2 BHIA4V0.5
RESISTANCE
DETECTION
Macro method E test
• Important screening tool for h VISA
• Utilizes larger volume 200μL of higher inoculum 2McF for prolonged
incubation 48h at 35 degree c
E test Glycopeptide Resistance detection
Double ended E test strip that contains gradient concentrations of
Vancomycin and Teicoplanin on each side
• Microcolonies
• Point of complete inhibition

• MET E TEST : Positive for hVISA : Vancomycin and Teicoplanin

MIC cut off >=8 µg/mL or Teicoplanin MIC cut off alone >=12 µg/mL
• GRD E TEST : MIC > =8 µg/mL
• Specificity 98-100% at 24h ; 82-95% at 48h

• Sensitivity 70-77% at 24h ; 93-94% at 48h

 Simplified Population Analysis Profile (PAP) or
Hiramatsu Screening Method
SCREENING AGAR
MHA5T2 BHIA4V 0.5

MEDIA MHA BHI AGAR

DRUG TEICOPLANIN VANCOMYCIN

CONCENTRATION 5µg/mL 4µg/mL

INOCULUM SIZE 2McF 0.5McF

INCUBATION Read at 24hr and 48hr Read at 24h and 48h

POSITIVE RESULT GROWTH OF COLONIES >=1 GROWTH OF COLONIES >=1

AT 48H
SENSITIVITY 65-79% 66%
SPECIFICITY 35-95% 82%
• 10 µL drop on 0.5McF isolate suspension is used to spot a 10-15mm
diameter of a BHI plate.

• >1colony : s/o VISA/VRSA

Population Analysis Profile Assay – PAP Assay

Population Analysis Profile area under curve PAP -AUC

Gold standard method for detection of hVISA in which the population

profile of the isolate is analyzed on agar plates containing a range of
Vancomycin concentrations.
• A standard inoculum of overnight broth culture
is plated on to BHI agar plates containing
increasing concentrations of Vancomycin 0.5-
4μg/ml .

• Plate counts are graphed to calculate the

proportion of cells that are Vancomycin
resistant with each dilution.

VSSA : PAP/AUC ratio of <0.9

hVISA : PAP/AUC ratio of >=0.9
Modified PAP
Differs by using a reference test as comparator.

Involves calculating area under the curve of the standard PAP graph and
comparing the result of the test organism reference strain such as
Mu3 strain (for hVISA ) and Mu50 strain (for VISA)
Reporting Vancomycin for Staphylococci

• Vancomycin-susceptible staphylococci should be reported following the

laboratory’s routine reporting protocols.

• Staphylococci not susceptible to vancomycin preliminary results should

be reported following routine reporting protocols.
• Any S. aureus with MICs ≥ 8 µg/mL should be sent to a referral
laboratory for confirmation.

• Public health authorities should be notified according to local

recommendations.
Reporting :

If test for hVISA is performed : “Detection of h VISA and avoid using

Vancomycin for therapy”

BUT, there is no standard guideline for detection of h VISA

High Level Mupirocin Resistance
Mupirocin (pseudomonic acid A) : The only approved antibiotic for
decolonisation of MRSA and methicillin-susceptible S. aureus (MSSA)
both in patients and healthcare personnel

HLMR indicated from nasal carriers or superficial wounds.

• Two types of resistance to Mupirocin

• Highlevel : plasmid-mediated mupA gene

• Low-level : chromosomal point mutations.

• The high-level mupirocin resistance phenotype can be transferred by

conjugation between different staphylococcal strains or species.
 High Level Mupirocin Resistance

Incubatio Additional
n testing
Test Organis Antimicrobial condition and
Test method m group Medium concentration s Incubation time Results reporting
Report
isolates
Examine carefully with
with no
transmitted light for
zone as
light growth within the
high-level
zone of inhibition.
35°C mupirocin
Disk 200-μg Standard disk 24 hours; read
S. ±2°C; resistant.
diffusio MHA mupirocin diffusion with transmitted No zone = high-level
aureus ambient Report any
n disk procedure light mupirocin resistance.
air zone of
inhibition
Any zone = the
as the
absence of high-level
absence of
mupirocin resistance.
high-level
resistance
 Antimicrob
Test ial Additional
metho Organism concentrat Inocul Incubation Incubation testing and
Test d group Medium ion um conditions length Results reporting
For single
256-μg/mL
Report growth in
well:
the 256-μg/mL
well as high-level
High Growth =
mupirocin
Level Single high-level
Broth resistant. Report
Mupir mupirocin 24 35°C ±2°C; mupirocin
Microd S aureus CAMHB 24h no growth in the
ocin 256-μg/mL hours ambient air resistance.
ilution 256-μg/mL well as
Resist well
the absence of
ance No growth =
high-level
the absence
resistance.
of high-level
mupirocin
resistance.
REPORTING
S .aureus with HMR: Avoid using Topical Mupirocin for the
decolonization of nasal carriers or superficial wounds”
• Recommended Vancomycin Testing in Staphylococcus : MIC based
test (BMD )

• VSSA can turn into VISA during course of prolonged therapy,

therefore repeat testing is warranted for subsequent isolate after 3
days.
Resistance Detection in
Enterococci
 VRE
• Five recognized phenotypes of vancomycin resistance, VanA, VanB, VanC, VanD, and
VanE
VanA Inducible
high-level resistance to vancomycin (MICs, ≥64
μg/ml) and teicoplanin (MICs, ≥16 μg/ml)

VanB Inducible
resistant to 4 to ≥1,000 μg/ml whereas susceptibility to
teicoplanin is retained.
VanC low-level
resistance to vancomycin (MICs, 4 to 32 μg/ml) and
are susceptible to teicoplanin.

E.casseliflavus and E. gallinarumintrinsic,

Vancomycin Resistant Enterococci
Intrinsic

• Low level resistance to Vancomycin

• Enterococcus gallinarum,E.casseliflavus,E.flavescens

Acquired

• Enterococci can become resistant to vancomycin by

acquisition of genetic information from another organism.
• E.Faecalis ,E.faecium

Genes : van A,B,C,D,E

 KB Disk
Broth dilution
diffusion

Vancomycin
Resistance MIC testing Agar dilution
Detection

Gradient
Screen Agar
Diffusion E test
Vancomycin Disk Diffusion 30μg ZONE DIAMETER

SUSCEPTIBLE >=17

INTERMEDIATE 15-16

RESISTANCE <=14
VANCOMYCIN MIC MIC BREAK POINTS

SUSCEPTIBLE <=4

INTERMEDIATE 8-16

RESISTANT >=32
Most enterococci are tolerant to the bactericidal activity of β-lactam and
glycopeptide antibiotics, bactericidal synergy between one of these antibiotics and
an aminoglycoside is needed to treat most serious enterococcal infections such as
endocarditis and meningitis
High Level Aminoglycoside Resistance
(HLAR) Screening
• For Enterococci Aminoglycosides may appear active in vitro, but not
effective clinically ,should not be reported as Susceptible.

• Aminoglycoside + Cell wall acting agents  Synergy – effective in

vivo.
High Level Aminoglycoside Resistance
(HLAR) Screening
• Performed for Enterococcus spp to determine the synergy of
aminoglycosides with cell wall acting agents

• Methods:KB-DD or MIC based methods

• Agents:
a. KB –DD: Gentamicin 120 μg or Streptomycin 300 μg disk
b. MIC : Gentamicin 500 μg /ml or Streptomycin 1000 μg /ml for
BMD and 2000 μg /ml for Agar dilution
• If an Enterococcus isolate is shown to have High level resistance to
Gentamicin, then the isolate is considered High level resistant to other
Aminoglycosides, except Streptomycin.
Detection of Resistance in
Pneumococci
 Disk diffusion

Detection of
Resistance in Broth dilution
Pneumococci

Agar Dilution
For Non meningitis isolates Susceptible Intermediate Resiatnce

Oxacillin 1μg (DD) >=20 - -

Pencillin (MIC) <=2 (CLSI) <=0.06μg /ml

(EUCAST)

<=19mm : Perform MIC testing ,then only report as Penicillin Resistance

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