ANTIBIOTIC SUSCEPTABILITY TESTING (Kirby Bauer Disk Diffusion test) PURPOSE: It is used to asses the antibiotic sensitivity of a certain bacterial isolate. SPECIMEN : Pure bacterial isolate from fresh culture plate. MATERIALS : 1. Nutrient broth for fastidious organisms or sterile saline for non fastidious organisms. 2. 0.5 Mc Farland standard for adjusting the turbidity of the inoculums. 3. Vortex mixer for suspension of the inoculum. 4. View box for comparison of broth with standard 5. Mueller-Hinton agar plates unsupplemented for non fastidious organisms or supplemented with RBCs in a concentration of 5% for fastidious organisms (90-mm diameter for seven disks; 150- 1
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ANTIBIOTIC SUSCEPTABILITY TESTING
(Kirby Bauer Disk Diffusion test)
PURPOSE:
It is used to asses the antibiotic sensitivity of a certain
bacterial isolate.
SPECIMEN :
Pure bacterial isolate from fresh culture plate.
MATERIALS :
1. Nutrient broth for fastidious organisms or sterile saline
for non fastidious organisms.
2. 0.5 Mc Farland standard for adjusting the turbidity of
the inoculums.
3. Vortex mixer for suspension of the inoculum.
4. View box for comparison of broth with standard
5. Mueller-Hinton agar plates unsupplemented for non
fastidious organisms or supplemented with RBCs in a
concentration of 5% for fastidious organisms (90-mm
diameter for seven disks; 150-mm diameter for a
maximum of 12 disks) from a lot that gives a
satisfactory quality control results. The PH must be 7.2
to 7.4, and the depth must be approximately 4 mm.
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6. Non-CO2 35- 37˚C incubator for non fastidious
organisms or 5% CO2 incubation for H.inf -
S.pneumoniae- N.meningititis
QUALITY CONTROL
Antibiotic discs for susceptibility testing are checked
weekly utilizing appropriate ATCC reference strains. In
addition, QC testing will be performed anytime when
antibiotic with a new lot number is used repeat the testing.
Document any corrective action in the QC log book. The
discs tested for QC must be the same discs used with the
patient specimens. Tolerance limits for antimicrobial potency
are based on CLSI guidelines. If the zone range limits are
exceed, the Lab Director must be immediately notified and
no sensitivity results will be reported.
E coli ATCC 25922Pseudomonas aeroginosae ATCC 27853S. aureus ATCC 29213
ALSO QC STRAINS FOR ESBL & FASTIDIOUS ORGANISMS MUST BE INCLUDED
QC ORGANISMS MAINTENANCE :
Avoid repeated subculture
Store stock isolates at -60C or below
Prepare working culture weekly & stored at -20C
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PROCEDURES
1. Preparation of inoculum :
a. With a sterile wire loop, touch the top of two to
five similar- appearing, well-isolated colonies on
an agar plate culture according to the size of
colonies as follows: large colonies as
citrobacter touch only the quarter of its size,
small colonies as strept touch five colonies,
while moderate sized colonies touch only
two colonies.
b. Emulsify them in 5mL of sterile physiological saline
or nutrient broth with the help of vortex.
c. The turbidity of the emulsification is adjusted to
0.5Mc Farland standard. Turbidity is matched
against a printed card or sheet of paper in a good
light.
d. Within 15 minutes of adjusting the turbidity of the
inoculums suspension, add the suspension to the
plate by pouring the suspension on the surface of
the agar plate, and then discard the excess in
waste container which contain a disinfectant
Replace the lid of the dish .Allow at least 5
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minutes but no longer than 15 minutes for the
surface to dry before adding the antibiotic disks
2. Testing of antibiotics:
a. Place the appropriate antimicrobial-impregnated
disks with specific concentration according to
(CLSI recommendation, age, pregnancy, inpatient
vs outpatient, type of specimens ) on the surface
of the agar, using forceps. Disks must be evenly
distributed on the agar so that they are no closer
than 25 mm from center to center and about 15
mm from the edge of the agar plate.
b. Gently tamp each disk down onto the agar to
provide uniform contact.
c. Within 15 minutes of applying the disks, invert the
plate and incubate it aerobically (ambient air) at
37˚C for 16-18 hours. Examine the plates after the
overnight incubation except for staph & strept up
to 24 hours
INTERPRETATION
With the use of a ruler or a template, the zones of
complete growth inhibition around each of the disks are
carefully measured to within the nearest millimeter; All 4
measurement are made by the unaided eye while viewing
the back of the petri dish with reflected light against a black,
non reflecting background. The plates should be viewed from
a directly vertical line of sight to avoid any parallax that may
result in misreading.
An interpretive correlate (susceptible, moderately
susceptible, intermediate or resistant) is provided by
reference to published CLSI guidelines.
LIMITATIONS
1- Do not move a disk once it has contacted the agar,
because some of the drug diffuses almost immediately.
2- Susceptibility plates prepared with blood must be
viewed from the agar surface and measurements made
with the cover of the Petri dish removed.
3- Zones that fall into the intermediate range should be
considered equivocal; if therapy with the drug is desired, a
dilution susceptibility test should be performed to clarify
the issue.
4- When testing staphylococci against methicillin or
oxacillin or enterococci against vancomycin, incubation
should be for 24 hours.
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5- Motile organisms such as Proteus mirabilis or P.vulgaris
may swarm when growing on agar surfaces, resulting in a
thin veil that may penetrate into the zones of inhibition
around antimicrobial agent susceptibility disks. This zone of
swarming should be ignored; the outer margin, which is
usually clearly outlined, should be measured. Similarly, with
sulfonamide disks, growth may not be completely inhibited
at the outer margin, resulting in a faint veil, where 80% or
more of the organisms are inhibited. The clear zone of ~
80% inhibition should be read as the zone diameter.
6- Presence of distinct colonies within the zone of inhibition
(2ry colonies) represent either mutant of the same species
that are more resistant to the antimicrobial agent than the
major portion of the bacterial strain being tested or the
culture is not pure and the separate colonies are of a
different species. If it is determined that the separate
colonies represent a variant of a mutant strain, the bacterial
species being tested must be considered resistant. If it is
determined to be a different species, return to the culture
Petri dish and realize whether it is a missed colony or a
contamination. If missed, do a separate antibiogram for the
isolate.
7- When there is overlapping between adjacent agent
zones, zones extend beyond the margin of the Petri dish
or oval(elliptical) zones;, the test must be repeated with
more careful placement of the antimicrobial agent disks
so that overlapping will not occur .When the plate is
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streaked poorly , this will lead to indistinct zones and the
test must be repeated
CHOICE OF ANTIBIOTICS IN ANTIBIOGRAM:
Drugs are listed by CLSI in 4 groups:
1- GROUP A: Testing & reporting against all isolate.
2- GROUP B: Testing when isolate is resistant to groupA.
3- GROUP C: Supplemental or alternative agent that can
be tested &reported in institutions that harbor resistant
strains.
4- GROUP U: Agents that should be tested & reported only
on isolates from urine.
5- Group O :
6- Group I :
Protocol of antibiotics choice in mic. lab
IN THE FIRST DAY GROUP A & B (GROUB 1) are tested in non urine isolates & (GROUP U) in urine isolates.
IN THE SECOND DAY
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GROUP C ( GROUP 2) for H.Infleuanza & Enterobacteriacae & antimicrobial combinations will be done .
IN THE THRID DAY Another combination will be tested in multi resistant strains
Antimicrobial agents with FDA clinical indication that should be considered for routine testing
Acinetobacter Fortum (CAZ) Tienam (IPM) or Meronam(MEM) Unasyn (SAM) Ciprofloxacin(CIP) or Levofloxacin(LEV)
Or ofloxacin ( OFX) Gentamycin (CN) or tobramycin (TOB) or
amikin (AK) SUTRIM ( SXT) Sulperazone (SCF) Cefotaxime (CTX) or Rocephine (CRO) Doxycycline (Do) or Tetracycline (TE) Tazocin (TZP)
N.B If oxacillin sensitive S.pneumoniae report blindly all penicillins & cephalosporins sensitive but if resistant MIC for 3rd generation cephalosoprins is mandatory
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Enterococci ( non urine) Staph ( non urine ) Penicillin(P) Ampicillin (AMP) Vancomycin (VA) CN 120µg (high level
screen) or Streptomycin Erythromycin or
Azithromycin Tetracycline ( TE)
Cefoxitin (Fox 30g) Penicillin (P) if sensitive
report all penicillins cephalospoines & carbapenems are sensitive approved by FDA
Sutrim (SXT) Clindamycin(Cd) test of MLS
resistance is recommended Azithromycin(AZM)or
Erythromycin (E) Vancomycin(VA) CIP or OFX or LEV in MSSA
only DO or TE Gentamycin ( CN) Caphalothin ( CF )
Enterococci( urine )
Ciprofloxacin(CIP) Levofloxacin Norfloxacin
Furadantin (F) Tetracycline(TE) P AMP Vancomycin
Staph (urine )
Norfloxacin(NOR) Ofloxacin(OFX) Levofloxacin FOX P VA Furadantin (F) Sutrim (SXT)
producing organism with 3rd generation cephalosporins
may result in clinical failure if infection is (outside the
urinary tract).
Testing of cephamycins is recommended in ESBL
producing isolates.
Cefpodoxime and ceftazidime have been proposed
as indictors of ESBL production as compared to
cefotaxime and ceftrioxone.
These enzymes can be induced by certain Abs,
AAs, or body fluids.
It is possible for one specimen to contain both
ESBL producing and non ESBL producing cells of the
same species. So, it must test several colonies for a
primary culture plate.
Latest guidelines recommended screening of ESBL
with a MIC 2mg/dL against cefpodoxime ceftazidime,
aztreonam, ceftaxime or ceftrixone.
Three indicators of ESBL:
An 8 fold reduction in MIC in the presence of
clavulonic acid by broth dilution method.
Potentiation of the inhibitor zone by clavulonic
acid >5mm in diameter of inhibition by disc
diffusion.
Disc approximation test by using of cefoxitin
(inducer) placed at a distance of 2.5cm from
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cephalosporin disc flattening of the zone of
inhibition of cephalosporin disc towards inducer
disc >1mm.
As regards treatment of ESBL carbapenens are the
most effective and reliable as they are highly
resistant to hydrolytic activity of all ESBL enzymes
due to trans-6-hydroxy ethyl group.
Meronam is the most active with MICs generally
lower than those of IPM (0.03-0.12mg/ml vs 0.06-
0.5mg/ml).
Also ESBL activity is inhibited by clavulonic acid,
the only infections that can be treated safely with
lactamase inhibitor are those involving the urinary
tract in which the concentration high enough to
counteract the hydrolytic activity of ESBL.
Clavulonic acid appears more efficient than
sulbactam it takes about eight times more to obtain
a protective similar to that by C.acid.
Plasmids responsible for ESBL production tend to be large and carry resistance to several agents an important limitation in the design of treatment. The most frequent co-resistance are aminoglycosides, flouroquinolones , TE, chloramphenicol and sutrim
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Issue No/Revision No:Ain Shams University
HospitalsIssue date:Revision date:Copy number:
Code No:Main LaboratoriesPage of
ANTIMICROBIAL COMBINATION BY DIFFUSION METHODS:
Disk approximation test :
Principle:
This method has been explored to assess primarily in a
qualitative fashion the interaction of antimicrobials as they
diffuse through agar plates seeded with a test organism.
Advantages:
Simple.
The use of readily available materials (discs and
Muller Hinton agar).
Disadvantages:
Qualitative method only.
Low sensitivity and specificity compared to dilution
methods.
i.e.: The results of this test may differ from results obtained when the same agents and organism are tested in liquid media.
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Procedure: This technique uses the same standard inoculums
and Muller-Hinton agar as a routine Bauer-Kirby
susceptibility test.
To assess possible interactions between two drugs
(A and B) disks containing these drugs are placed on a
plate that has been inoculated with a tested organism.
The distance by which the disks are separated
may be varied, but it should generally be equal to or
slightly greater than the sum of the radii of the zones of
inhibition of the drugs when examined alone (mostly
15mm from centre to centre).ONLY FIVE COMBINATION
ARE TESTED IN THE 100 mm PLATE
After overnight incubation (16-18hrs) at 37C the
plate are ready for examination.
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Issue No/Revision No:Urine Microbiological
ExaminationAin Shams University
Hospitals Issue date:Revision date:Copy number:
Code No:Main LaboratoriesPage of
Example of antibiotic combinations used for multi resistant organisms "by Dilution methods":1. Pseudomonas:
Bactericidal:
Ciprofloxacin and tienam (CIP and IPM).
Ciprofloxacin and Amikin (CIP and AK).
Ciprofloxacin and Azactam (CIP and ATM).
Ciprofloxacin and Fortum (CIP and CAZ).
Levofloxacin and Maxipime (LEV and FEP).
Ciprofloxacin and Maxipime (CIP and FEP).
Levofloxacin and Meronam (LEV and MEM).
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Ciprofloxacin and Tazocin (CIP and TZP).
Levofloxacin and Tazocin (LEV and TZP).
Levofloxacin and Gentamycin (LEV and CN).
Tazocin and Gentamycin (TZP and CN).important
Tazocin and Tienam (TZP and IPM).
Bacteriostatic:
Augmentine and Ampicillin (AMC and AMP).not used
Vanocomycin and Carbencillin (VA and Pip).not used
Azactam and Maxipime (ATM and FEP).
2. Acinetobacter:
Bactericidal :
Doxycyclin and Amikin (Do and AK).
Ciprofloxacin and Fortum (CIP and CAZ).
Ciprofloxacin and Meranam (CIP and MEM).
Ciprofloxacin and Azactam (CIP and ATM).
Tazocin and Gentamycin (TZP and CN).
Ciprofloxacin and Tazocin (CIP and TZP).
Bacteriostatic :
Tienam and Amikin (IPM and AK).
Unasyn and Amikin (SAM and AK).
3. Enterobacteriaceae:
o Tazocin and Amikin or Gentamycin (TZP and AK or
CN).
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o Cefotaxim and Amikin or Gentamycin (CTX and AK
or CN).
o Azactam and Tienam (ATM and APM).
o Azactam and Maxipime (ATM and FEP).
o Ceftazidime and Oflaxocin (CTZ and OFO).
o Cefoxitin and Amikin (FOX and AK).
o Ciprofloxacin and Fortum (CIP and CAZ).
o Ciprofloxacin and Tazocin (CIP and TZP).
4. Proteus:
o Tazocin and Amikin (TZP and AK).
o Tienam and Amikin (IPM and AK).
5. Enteroccoci:
.lactam (penicillin) and amino glycoside.
(gentamycin )
Glycopeptide (VA or TEC) and aminoglycoside.
Teinam and Teicoplanin (IPM and TEC).
Tazocin and Gentamycin (TZP and CN).
Tazocin and Ciprofloxacin (TZP and CIP).
Glycopeptide and .lactam (TEC and P).
Ciproflxocin and Vancomycin or Penicillin (CIP and