Antimicrobial Susceptibility Testing finale 2.pdf

bacteriology

1. CALVIN C. BODOSO‚ RMT
Antimicrobial
Susceptibility
Testing

2. INTRODUCTION
AntimicrobialSusceptibilityTestingisperformed
onbacteriaisolatedfromclinicalspecimensto
determinetheantibioticsthatmightbeeffectivein
treatinginfectionscausedbybacteria.
O -
=
>
=>
-
*
=
-

3. MINIMUM INHIBITORY CONCENTRATION
(MIC)
Lowestconcentrationofanantimicrobialagentthat
visiblyinhibitsthegrowthoftheorganismbeingtested.
Theabilityofanantimicrobialagenttoinhibitthe
multiplicationofanorganismismeasuredbytheMIC.
minimum
inhiditiy
concentration
(MIC)
lowest
a
Minimum
①
=
O
=
-
&
-
-
E
-

4. MINIMAL BACTERICIDAL
CONCENTRATION (MBC)
Thelowestconcentrationofantimicrobialagentthatkills
99.9%ofthetestbacteria.Thereductioniscalculatedin
CFU/mLcomparedwiththenegativecontrol.
MinimalEntericidal
concentration
O=>
z
=>
CFu/mL
(compardwl
-
c)
-
-
mini
num
whi
-
S
thereconvention
I
a
-
muimal
backsi
(MBC)

5. DILUTION METHODS
Dilution
①againDilutionmethod
e
InteDilution
buthic

6. Agar Dilution Method
Inthismethod‚aseriesofagarplatesarepreparedwith
differentconcentrationsoftheantimicrobialagentbeing
tested.Theseplatesareinoculatedwithastandardized
amountofthemicroorganismbeingstudied.Afteran
incubationperiod‚theplatesareexaminedforgrowth.The
lowestconcentrationoftheantimicrobialagentthat
completelyinhibitsthegrowthofthemicroorganismis
determinedastheminimuminhibitoryconcentration(MIC).
FinalInoculum:1x10^4CFU/mL
1410
"cEulmAbmO
=>=>
⑤
A
-IX/04
(n/m
O
B
-
5X105cn/m/

7. Broth Dilution Method
Inthismethod‚aseriesofagarplatesarepreparedwith
differentconcentrationsoftheantimicrobialagentbeing
tested.Theseplatesareinoculatedwithastandardized
amountofthemicroorganismbeingstudied.Afteran
incubationperiod‚theplatesareexaminedforgrowth.The
lowestconcentrationoftheantimicrobialagentthat
completelyinhibitsthegrowthofthemicroorganismis
determinedastheminimuminhibitoryconcentration(MIC).
FinalInoculum:5x10^5CFU/mL
ANOYAn
⑧

8. Broth Dilution Method
AmLMAB-2-10
,
11
,
13
2mL
MtB-12
controlAm200ngIm)AS -1
,
2
,
13Il-monlum
control
12-brothcontrol Thrath
2
-
11
S13
(ent)
12
(inc)
+
%03m
~
↓L
x
1-11
1
,
2
,
13
370
1-11 2-11
,
13
4/2
u
1,
2
,
13
two-folddilutions
-
2
&
MASI
3-18 1m/momlum(1X10CFNIm()-1-11
1-11

9. Preparation of Inoculum

10. Preparation of Inoculum
Purecoloniesmustbeutilized
Standardizedinoculum
YoungandPurecolonies>>4-5colonies>>
TSB/NSS/Distilledwater>>Incubation>>Compareto
the0.5McFarlandStandard
%
-
5colonies pureclonies
"young
pure
cl(4-52)
-
6
TND
O
Tryptic
son
brath
&
-0
.
5 McFarland
monies
NS
-
-
Mutation
Disheld
wate
&
compare
0
.
5
McFarland
-
sundard

11. Preparation of Inoculum
Thestandardmethodistocomparetheturbidityofthe
testliquidmediumwiththatofastandardthat
representsaknownnumberofbacteriainsuspension.
Tubeswithvaryingconcentrationsofthischemicalwere
developedbyMcFarlandtoapproximatenumbersof
bacteriainsolutionsofequalturbidity.
-
·
StandardmethodA
batt
O
z
O
=>
- - -
-
E
-
-
E 3

12. McFarland Standards
McFarland0.5standardisthemostfrequentlyused:
0.5mLof1.175%BariumChloride(BaCl)
99.5mLof1%v/vSulfuricAcid(H2SO4)
Themixtureprovidesaturbiditycomparabletothatofa
bacterialsuspensioncontainingapproximately1.5×108
CFU/mL.
0
.
51
.
15BC↑
mynsearch
0
.
5
-
commonly
me
"¥
sa
S
Gradeunad
99
.
5
17
-0
.
5 m)g
1
.
AJInBarn
i
&
-
99
.
5m
&1%viuSulfuric
and(2004)
O
bactical
suspension
-
1
.
5X108cFulm
=
-
=
-
minsland
0
.
5
standard
0
.
5m)
1
.
175Back
0
.
511753B
1
.
5
x
108
~
g 9951
%SA-99
.
Tm/g/%virHe did

13. Diffusion Methods
-
Diffusion

14. Kirby-Bauer Disk Diffusion Method
Standardizedinoculumispreparedasdescribedandis
swabbedoverthesurfaceoftheagarplate.
Kisty-Bauer
DickDiffusionmethodO
Kirby
-
BauerDDM
-
- =

15. Kirby-Bauer Disk Diffusion Method
CLSI requirementscovers:diskconcentrations‚
standardizationofmedia‚formula‚pH‚agardepth‚inoculums
density‚temperature‚zonesizes‚interpretativetables‚and
referencestrainsofbacteriaforcontrols.
⑳
=
siTIR

16. Kirby-Bauer Disk Diffusion Method
CultureMedium:
Mueller-HintonAgar(MHA)
1.pH:
2.Depth:
3.Cationconcentration:
•Calcium:25mg/L
•Magnesium:12.5mg/mL
KBDDM
m
-
MHA
pH-7
.
2-7
.
4
7
.
8-7
.
4(4--standard)n -3-5mm(4)
3
.
5
mm
ce-2a-25mg
7
.
2
-
+4
&my-12
.
SingImd
-
25igl
3
.
0
(4)
i
-
12
.
5
myInt

17. Kirby-Bauer Disk Diffusion Method
PreparationoftheMHA:
Dryingfor_______at_______beforestorage
Storage:__________temperatureformaximumof__weeks
Moisture:
ThesurfaceoftheMHAmustbemoist.
Afterinoculationandbeforeapplyingtheantimicrobialdisks‚
allowtheexcessmoisturetobeabsorbed:
_____mins‚butnotmorethan____mins
Bring
-
10-30mins
(35
°
C)
10-30mins35
%
Repignator L
(2-802)
Storage
-
Religerator(2-8
:
c)E
Imax
:
I
weeks
=>
-
3-5
-
15
>
3-5mins
>
X75mis
.

18. Kirby-Bauer Disk Diffusion Method
PreparationoftheMHA:
16-18
35
°
C;
room
ai
20
-
24strep
Neiss
SHN
A
=>
35
%
#
=
*
O 5-702
HTM
6
6(A
+
S

19. Kirby-Bauer Disk Diffusion Method
Antibioticdisks:
Nitrocellulosefilterpaper
Diameterof___mm
Disksplacements:
<6disksshouldbeplacedona___mmplates
<12disksshouldbeplacedona___mmplates
Duringincubation:
DoNOTstackmorethan5plateshigh
antibioticdisks
6
"¥
withclub
so
fitte
paper
D
=
Emm
100
<
6
=
100mm
150 4)
2
=
150mm
E
6-100mus
12-150mm
=
0
=

20. Kirby-Bauer Disk Diffusion Method
B
A
C
10-15mmfromtheedgeof
theplate
24mm:spaceofeach
antimicrobialdisk
10-15mm
=
from
Edge
O
"¥
-&
-
24mm
=
spmeeach

21. Kirby-Bauer Disk Diffusion Method
Procedure:
1.PreparationofInoculum
2.Selectionofantibioticdiscs
3.InoculateMHagarusingpreparedinoculum
•Streakingsterileswabacrosstheentiresurface
•Repeattwice:turningtheplate60degreesbetweeneach
streaking
•After3
rd
streaking‚rimsidesoftheagar
Splate
thing
680
-
-O
-
-
Er
after:
him
sidesofagai

22. Kirby-Bauer Disk Diffusion Method
Procedure:
4.DryingoftheInoculatedMHA
5.Applicationofantibioticdiscs
6.Incubation
•Inverttheplates
•35-37Cfor16-18hrs
7.ReadingandInterpretationofresults
initation
it
plates
-
35-372(16-10hrs)
-hist
plates
35-370(16-18
hours)

23. SUSCEPTIBILITY AND RESISTANCE
Susceptible-indicatesthatthepatientmostlikelywill
respondtotreatmentwiththeantimicrobialagent.
Resistant-indicatesthatthetreatmentwiththeagent
willmostlikelyfail.
Intermediate-meansthatahighdose(ifpossiblewiththe
agent)maybenecessaryforasuccessfultreatment
outcome.
S=
TD
SRI
Susceptible
-
Respond
-
SRI Resistant
-
Fail
Intermediate
-
high
dosage

24. Reading and InterpretationofResults
Beforeresultswithindividualantimicrobialagentdisksare
read‚theplateisexaminedtoconfirmthataconfluentlawnof
growthhasbeenobtained.
Mixedculturesareevidentthroughtheappearanceof
differentcolonymorphologiesscatteredthroughoutthelawn
ofbacteria.
- -
-
mixed
cultures
-
appearance
g
&
affrentwang
morphologies

25. Reading and InterpretationofResults
Adarkbackgroundandreflectedlightareusedtoexamine
diskdiffusiondiameters.Arulerorcalipercanbeusedto
measurethezonediametersforeachantimicrobialagent.
dark
background
an
reflectedlight
DDD
-za/caliper--

26. Gradient Method

27. Gradient Method: E-Test / Epsilometer
Test
•Utilizes MHA plus a plastic strip impregnated with
antibiotics on one side and the other side with the label of
concentration.
•Inoculate organism to MHA then place the strip
•Incubate for 24 hours.
•Observe for ZOI(MIC)
Epsitimatel/E-test
①
MAA
MAntibioticsconcentration
-
Gradient
willd -G-test/Epsidometerlast

28. Gradient Method:
E-Test / EpsilometerTest

29. Automation

30. AUTOMATION
-
Tartidametery
=
um
Redox-indicatorB
VITic1/213[
MSNP
BDPAMS0
= 2
Resnzusin
Almar
R
=
G
,
P
A
=
B
,
P

31. PnaggingResistant
Staph
SuCind
a
myis
basmay
ERM
gene
S
"¥
modifiedKinky-Bauer

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References: