Antibiotic

Sensitivity
Testing
Margie A. Morgan, PhD
MT(ASCP), D(ABMM)
 Antibiotic Classes
Penicillins Anti- Staph Penicillins
Penicillin Nafcillin
Amoxicillin Oxacillin
Ampicillin Cloxacillin
Amp/Clavulanate Dicloxacillin
Amp/Sulbactam These antibiotics and
Anti-Pseudomonal those listed on the
Penicillins next slide have a beta
Piperacillin/Tazobacta lactam ring structure
m aka beta lactam
Ticarcillin/Clavulanate antibiotics
 Antibiotic Classes (2)
Carbapenems Cephalosporins
Imipenem
First generation
Cefazolin
Meropenem Second generation
Ertapenem Cefotetan
Dorapenem Cefoxitin Cefuroxime
Third generation
Cefotaxime Ceftriazone
Monobactam Ceftazidime
Cefpodoxime
Aztreonam Fourth generation
Cefepime
 Antibiotic Classes (3)
Fluoroquinolones Macrolides
Ciprofloxacin Azithromycin
Levofloxacin Clarythromycin
Moxifloxacin Erthromycin
Aminoglycosides Tetracyclines
Gentamicin Tetracycline
Tobramycin Minocycline
Amikacin Doxycycline
Trimethoprim/ Lincosamide
sulfamethozaxole Chloramphenicol
 The Four Major
mechanisms of antibiotic
resistance
Enzymatic cleavage leads to inactivation of antibiotic

Beta lactam and aminoglycoside antibiotics

Active Beta lactamases and amino-glycoside modifying
enzymes
Altered receptors/binding proteins preventing
attachment of antibiotics to the bacterial surface
Altered Penicillin binding proteins
Strep pneumonia resistance to penicillin
MRSA resistance to methicillin
Altered permeability/influx and efflux pumps stopping
passage through porins gram negative bacilli
Pseudomonas resistance to amino-glycosides
Bypass of a metabolic block/metabolic block imposed
by antibiotic
Enterococcus resistance to TMP/SXT
 The Rules for
Susceptibility Testing
1. CLSI Clinical Laboratory Standards
Institute Approved standards for the
testing & reporting of susceptibility
results/ updated yearly
1. Charts with appropriate antibiotics to test
2. How to interpret the laboratory results
3. QC standards and proper testing procedures
2. Susceptibility Tests are tests of bacterial
stasis not killing
 Methods/Bacteria in
METHODS
Review
1. Kirby Bauer disk diffusion
2. E Test Strip Minimum inhibitory concentration (MIC )
3. Broth dilution Minimum inhibitory concentration (MIC)
4. Beta lactamase enzyme detection
RESISTANT BACTERIA IN THE NEWS
MRSA methicillin resistant Staphylococcus aureus
VRE vancomycin resistant enterococcus
ESBL Extended Spectrum Beta Lactamase Gram neg
rods
KPC Klebsiella pneumonia Carbapenemase or CRE
(Carbapenamase Resistant Enterics)
Streptococcus pneumonia
Neisseria gonorrhoeae
 Preparation of Bacteria
for all Susceptibility
Methods
Requires pure culture of one organism only
Log phase growth of bacteria - 16-24 hrs old
Standardized suspension of bacteria must be
prepared using:
O.5 McFarland Standard Barium sulfate solution that
equals the turbidity of @ 10 8 bacteria/ml
Alternative method use spectrophotometer

Incubation of tests are at 35 *C in room air (some

require CO2) for 18- 24 hrs
This is a
0.5 McFarland Standard
which is a turbidity
standard made from
Barium sulfate the
turbidity is equal to
10 8 CFU/ml bacteria
 Quality Control for all
methods
Before you can test patients: Must test multiple QC strains for
20 consecutive days or 3 replicates for 5 days plan. This is to
make sure you can perform the tests correctly.
The QC values must be within CLSI established limits using
ATCC strains of organisms (American Type Culture
Collection) If QC strains are within limits - you can then do
Weekly quality control on all lots of cards, disks, plates in use
Data must be recorded and reviewed monthly by
supervisor
If weekly QC results are out of control:
Immediately repeat/ inform supervisor

If repeat is OK continue routine testing

If repeat is NOT OK must investigate/document/ repeat 5

times to start routine testing. All repeats must be in
control
 Agar Disk Diffusion (Kirby
Bauer Method)
Procedure: Qualitative Susceptibility method
Mueller Hinton agar with or without blood
150 mm plate diameter
4mm in depth
Agar specifically balanced in Ca+ and Mg+,
if the ions are too high % amino-glycosides test falsely resistant,
if the ions too low % falsely susceptible amino-glycoside results
Streak bacteria on plate with cotton tipped
swab
Apply 6mm paper disks that contains single
antibiotic
Incubate for 16-24 hrs at 35*C
Measure zone of diameter of inhibition of growth
(mm)
Kirby Bauer
disk

Dispenser
Each
Cartridge is
a separate
Kirby Bauer
Disk Diffusion

is c onsidered
ins i de a zone
Growth
e
resistanc

Measure diameter of the

Measure diameter in mm
Zone of across
inhibition in center
mm of the
Disk for each antibiotic disk

Colonies growing into zones is considered

Resistant to that antibiotic
Double zoning - measure the
inside zone
Watch out! The sensi could
Be mixed NEVER read a mixed
sensi!

Proteus swarms into zone and is

 Kirby Bauer (KB)
Concentration gradient created with the
diffusing antibiotic and the increasing number
of bacteria growing on the agar, this
determines the zone of inhibition around disk.
CLSI charts used to interpret the measured
zone sizes as Sensitive, Intermediate or
Resistant
Cannot directly compare zone sizes between
antibiotics
ex: ZID of 21mm zone size is as sensitive as a GM of
14mm - zone sizes differ for organism/antibiotic
combinations
Regression analysis can be used to calculate MIC
value related to KB zone size
 E Test
Quantitative MIC Susceptibility
Calibrated plastic strips impregnated with
one antibiotic/concentration gradient
(mcg/ml) embedded in plastic / carefully
placed on the agar surface
Gradient created as antibiotic diffuses into
agar in an elliptical shape
MIC (minimum inhibitory concentration) is
where the ellipse ends on the plastic strip
Useful for any organism but a method of
choice for slow growing fastidious organisms
E Test method

E test for Strep pneumonia

E test method
susceptibility
High concentration
MIC value

MIC value
Susceptibility result =
Where growth crosses
The plastic strip
Low
concentration
 Broth Dilution
Quantitative Susceptibility Method

Bacteria inoculum: 0.5 McFarland

standard further diluted to 5x105
organisms /ml in broth

Suspension is inoculated into tubes

or micro titer trays containing
growth medium and known 2 fold
dilutions (mcg/ml) of antibiotics
Doubling dilution MIC Tray
Each row has one antibiotic in >=
4 dilutions
 Broth Dilution
Definitions
MIC = lowest concentration of antibiotic inhibiting
growth
2 fold dilutions: 1 2 4 8 16 32 64 128 mcg/ml
Growth No growth
MIC = 8 mcg/ml
MBC - Minimum bactericidal concentration determined
by the subculture of the contents of the wells that show no
growth to solid agar - the lowest concentration of
antibiotic that kills 99.9% of original inoculum is the MBC.
8 16 32 64 128 32mcg/ml = MBC
Growth No Growth
Antibiotic tolerance - MBC/MIC ratio >=32
MBC = 128 MIC= 2 128/2= 64 Tolerance
 Siemens Microscan walkawa
AST system

BD Phoenix AST system

Biomerieux Vitek2 AST

 Disk test for Beta
lactamase Detection

(Cefinase Test)
Add bacteria to filter paper impregnated with Nitrocefin
(yellow colored/chromogenic cephalosporin)
Incubate at room temp (@ 1 minute) and observe for
color change from yellow to red

Positive result is color change to red

Bacteria beta lactamase enzyme breaks down the beta lactam ring of
Nitocefin to produce a red end product
Detects resistance to
Ampicillin/Penicillin/Cephalosporins in Haemophilus,
Neisseria gonorrhoea , Moraxella catarrhalis,
Enterococcus, and anaerobic gram negative rods
This test does NOT detect Extended Spectrum Beta
Lactamase enzyme produced by enteric gram negative
rods
Beta lactamase detection
tidbits
Haemophilus influenza
In US, approx 28% are beta lactamase
producers therefore, resistant to Ampicillin
Bacteroides fragilis group
Primary resistance mechanism is beta
lactamase production
>95% of strains are resistant to Pencillin

Moraxella catarrhalis
>90% beta lactamase positive /Ampicillin
resistant
 Methicillin Resistant
Staph aureus (MRSA)
In the laboratory you test oxacillin (OX) because it is
more stable for testing than methicillin
If OX is resistant the S. aureus is reported as a MRSA
Cefoxitin resistance testing is also a very reliable and
a preferred way to confirm a S. aureus to be a MRSA
All cephalosporin antibiotics are reported as resistant
when reporting results for MRSA and should never be
used for therapy
Resistance mechanism is by penicillin binding
proteins (PBP)
PBPs bind penicillin and related antibiotics
The binding prevents disruption of the peptidoglycan synthesis
in the Staph aureus cell wall
PBP are produced by the mecA gene
Methods to Detect Methicillin and
Oxacillin Resistance
Oxacillin is no longer
suggested by CLSI for
detecting MRSA
Oxacillin KB= resistant

Newer and more

sensitive
method to
screen for MRSA
is using a
Cefoxitin KB to
determine
Methicillin
resistance
 Clindamycin Induction
Test
Also known as the D test
This test accurately determines if Staph aureus,
including MRSA, is susceptible to Clindamycin
During antibiotic therapy, S aureus isolates
resistant to Erythromycin possess enzymes that
can be induced to make the S. aureus also
resistant to Clindamycin
Kirby Bauer zone around Clindamycin will be
blunted to form a D if Clindamycin can be
induced by Erythromycin to be resistant so
called INDUCIBLE RESISTANCE. Clindamycin
should be reported as resistant by clindamycin
induction and not used for therapy. Clinda is
often used to treat skin infections.
D test Positive
Blunt D shaped CC
zone
Clindamycin has
inducible
D test Negative
resistance
Round CC zone
 Enterococcus
All naturally resistant to:
Cephalosporins

Clindamycin

Trimethoprim/sulfamethoxazole

Synergistic antibiotic therapy can be important

for the treatment of Enterococcus
Ampicillin plus Gentamicin is synergistic and
often used to increase the killing potential
Important for endocarditis therapy
 Vancomycin Resistant
Enterococcus (VRE)
Acquired resistance to vancomycin
Plasmid mediated vanA associtated with E. faecium
Plasmic mediated vanB associated with E. faecalis
Not difficult to detect vancomycin resistance easily
detected by KB, automated AST systems, and Etest
methods
Drugs of choice if VRE detected
Linezolid
Synercid for E. faecium only
Major epidemiology issues!!
Rectal colonization can contaminatie the environment
and lead to transmission to patients
Most infections related to ICU stays and long
hospitalizations
Not virulent but problematic in immune suppressed
 Extended Spectrum Beta
Lactamase
[ESBL]
Enzymes that are produced by Gram negative bacteria
Confer resistance to Cephalosporins, Penicillins and Monobactam
(Aztreonam) by opening the beta lactam ring inactivating the
antibiotic
Cannot attack cephamycins (cefoxitin, cefotetan) or the
carbapenems (imipenem, meropenem, ertapenem, doripenem)
Generally susceptible to beta-lactamase inhibitors (tazobactam)
Plasmid mediated TEM, SHV, CTX-M beta lactamases are
the most common
Therapy for ESBL producing gram negative rods:
Carbapenems: Imipenem, Meropenem, Doripenem, Ertapenem
Piperacillin/Tazobactam Tazobactam blocks beta lactamase
action
 ESBL Susceptibility
Pattern
Escherichia coli ESBL Positive
Ampicillin R
Cefazolin R
Gentamicin R
Cefotetan S (Cephamycins are not
cephalosporins)
Cefotaxime R
Ceftazidime R
Cefpodoxime R
Piperacillin R
Pip/Tazobactam S (Tazobactam is a beta lactam
blocker)
Imipenem S (Carbapenem)
 Detecting ESBL in the
laboratory
Suggested method:
Cephalosporin MIC values and KB zone sizes
established by CLSI to safely detect ESBL
activity
New breakpoints are one to three doubling
dilutions lower than breakpoints and KB zones
for susceptible that were used for decades
These values were lowered to aid laboratories
in the correct detection of ESBL
Molecular testing needed for confirmation
beyond the scope of most clinical
laboratories
 The OLD method to detect
ESBL
[double disk test]
Test the susceptibility of a GNR against
a cephalosporin
Compare this zone size (KB) or the
MIC value with the same GNR tested
against the cephalosporin plus
clavulanic acid (a beta lactam blocker)
If the zone size or MIC is considerably
smaller with the cephalosporin alone,
you have evidence for ESBL production
by the GNR being tested
Positive ESBL Double
Disk Test
Resistant
Ceftazidime
Resistant
Cefotaxime

Susceptible
Cefotaxime plus
Susceptible Clavulinic acid
Ceftazidime plus
Clavulinic acid
 Why all the fuss?
Gram neg rods with ESBL phenotypes
>=10%
Left with limited treatment options
/Carbapenems such as Imipenem
Risk factors for development of colonization
or infection
Long hospital stay particularly in the ICU
Central lines
Issues with the intestine
Long term care facility
Ventilator assistance
 Carbapenemases
Carbapenem antibiotics have an important role they
retain therapeutic activity against resistant ESBL gram
negative rods
When carbapenem antibiotics are also inactivated
headed toward extreme resistance
Carbapenem-hydrolyzing-beta-lactamases confer
resistance to a broad spectrum of beta lactamase
substrates including carbapenems (CRE
Carbapenemase Resistant Enterics)
Two CRE are getting the most attention:
KPC Klebsiella pneumoniae carbapenemase is the most
common in the USA
NDM-1 New Delhi metallo-beta-lactamase has received much
press. Recognized in 2009. Resistance determinants are
numerous and great concern about its spread.
 Carbapenemase
producing GNR
Risk Factors
Acquisition of genes from other bacteria while
on very broad spectrum antimicrobial therapy
Severe illness
Mechanical ventilation
Organ transplantation
Medical care in India or Pakistan (NDM-1)
Can carry as normal intestinal flora
(colonization)
Fatality rates in infections can reach 50%
Carbapenemase Resistant Enterics
(CRE)
LaboratoryMost
Testing
sensitive screen and the first
Carbapenem to show subtle
resistance by increase in MIC value is
Ertapenem. However. most
Carbapenems will show elevated
MICs in the resistance range.

This class can be difficult to detect.

MIC values can be at the
breakpoint for resistance

Polymyxins (Colistin and Polymyxin B)

used for therapy. This class is toxic
 OLD Method to Detect
CRE/KPC/NDM-1
Modified Hodge Test
Observe growth on this
plate:
Background is lawn of E. Lawn of E. coli
coli
Test GNR streaks hug the Streaks of test
GNR
Imipenem disc in the
middle of the plate. (A, B,
C , D and E)

Hodge Test Positive = A

CRE positive
Shoulder effect around
streak of test organism

Negative = B
No shoulder effect
 Streptococcus pneumonia
and resistance to Penicillin
(PEN)
Two step resistance testing can be used
Step 1 - Oxacillin KB disk testing
If resistant to Oxacillin possible resistance to
PEN
Step 2 -Must confirm PEN resistance by MIC
test method
MIC value determines susceptibility or resistance

Best most direct method one step- Perform a

Penicillin MIC test by E Test or broth dilution
[cannot use KB to test Penicillin]
 Strep pneumonia MIC
values
Penicillin MIC values CLSI has set
interpretation depending on site of
infection
CSF Blood/Resp
Sensitive <=0.06 mcg/ml <=2 mcg/ml
Intermediate 0.12 - 1 mcg/ml <=4
Resistant >= 2 mcg/ml >=8
High level PEN resistance is uncommon,
usually <= 10%
If resistant to Penicillin antibiotics of choice
become Cefotaxime, vancomycin or a
quinolone
 Neisseria gonorrhoeae
(GC)
Increasing resistance of GC over last two decades
1980s beta lactamase producing strains emerged
eliminating Penicillin for therapy
By 2000, the quinolones were resistant due to the
acquisition of mutations that altered the binding sites
Currently Cephalosporins (Ceftriaxone & Cefixime) are
the mainstay for therapy in the US however
resistance to these antibiotics are becoming common in
Asia.
Resistance due to Penicillin Binding Proteins (PBP) and over
production of efflux pump
Detection of resistance in the USA could be
problematic due to our reliance on amplification testing
for STD diagnosis