This document discusses antibiotic sensitivity testing and provides updates from 2017. It describes various antibiotic classes and the major mechanisms of antibiotic resistance. It then covers the key methods for testing antibiotic sensitivity, including disk diffusion, E test, and broth dilution. Quality control and preparation of bacteria are also outlined. The document concludes by discussing specific resistant bacteria such as MRSA, ESBLs, and mechanisms of beta-lactamase production.

1. Antibiotic SensitivityAntibiotic Sensitivity
Testing 2017 UpdateTesting 2017 Update
Margie A. Morgan, PhD, MT(ASCP), D(ABMM)Margie A. Morgan, PhD, MT(ASCP), D(ABMM)

2. Antibiotic Classes
Penicillins
◦ Penicillin
◦ Amoxicillin
◦ Ampicillin
◦ Amp/Clavulanate
◦ Amp/Sulbactam
Anti-Pseudomonal Penicillins
◦ Piperacillin/Tazobactam
◦ Ticarcillin/Clavulanate
Anti- Staph Penicillins
◦ Nafcillin
◦ Oxacillin
◦ Cloxacillin
◦ Dicloxacillin
These antibiotics and those listed
on the next slide have a beta
lactam ring structure – aka beta
lactam antibiotics

3. Antibiotic Classes (2)
Carbapenems
◦ Imipenem
◦ Meropenem
◦ Ertapenem
◦ Dorapenem
Monobactam
◦ Aztreonam
Cephalosporins
◦ First generation
◦ Cefazolin
◦ Second generation
◦ Cefotetan
◦ Cefoxitin Cefuroxime
◦ Third generation
◦ Cefotaxime Ceftriazone
◦ Ceftazidime Cefpodoxime
◦ Fourth generation
◦ Cefepime Ceftaroline

4. Antibiotic Classes (3)
Fluoroquinolones
◦ Ciprofloxacin
◦ Levofloxacin
◦ Moxifloxacin
Aminoglycosides
◦ Gentamicin
◦ Tobramycin
◦ Amikacin
Trimethoprim/ sulfamethozaxole
Macrolides
◦ Azithromycin
◦ Clarythromycin
◦ Erthromycin
Tetracyclines
◦ Tetracycline
◦ Minocycline
◦ Doxycycline
Lincosamide
◦ Chloramphenicol

5. The Four Major mechanismsThe Four Major mechanisms
of antibiotic resistanceof antibiotic resistance
Enzymatic cleavageEnzymatic cleavage leads to inactivation of antibioticleads to inactivation of antibiotic
◦ Active Beta lactamases and amino-glycoside modifying enzymesActive Beta lactamases and amino-glycoside modifying enzymes
Altered receptorsAltered receptors/binding proteins preventing attachment of antibiotics/binding proteins preventing attachment of antibiotics
to the bacterial surfaceto the bacterial surface
◦ Penicillin binding proteins (PBPs)Penicillin binding proteins (PBPs)
◦ Mechanism for Strep pneumoniae resistance to penicillin and MRSAMechanism for Strep pneumoniae resistance to penicillin and MRSA
resistance to methicillinresistance to methicillin
Altered permeabilityAltered permeability/influx and efflux pumps stopping passage through/influx and efflux pumps stopping passage through
porins – gram negative bacilliporins – gram negative bacilli
◦ Pseudomonas aeruginosa resistance to amino-glycosidesPseudomonas aeruginosa resistance to amino-glycosides
Bypass of a metabolic blockBypass of a metabolic block/metabolic block imposed by antibiotic/metabolic block imposed by antibiotic
◦ Enterococcus resistance to TMP/SXTEnterococcus resistance to TMP/SXT

6. The Rules for SusceptibilityThe Rules for Susceptibility
TestingTesting
CLSICLSI – Clinical Laboratory Standards Institute Approved standards for– Clinical Laboratory Standards Institute Approved standards for
the testing & reporting of susceptibility results/ updated yearlythe testing & reporting of susceptibility results/ updated yearly
1.1. Charts with appropriate antibiotics to testCharts with appropriate antibiotics to test
2.2. How to interpret the laboratory resultsHow to interpret the laboratory results
3.3. QC standards and proper testing proceduresQC standards and proper testing procedures

7. Methods/Bacteria inMethods/Bacteria in
ReviewReview
Susceptibility testing methodsSusceptibility testing methods
1. Kirby Bauer disk diffusion (KB)1. Kirby Bauer disk diffusion (KB)
2. E Test Strip Minimum inhibitory concentration (MIC )2. E Test Strip Minimum inhibitory concentration (MIC )
3. Broth dilution Minimum inhibitory concentration (MIC)3. Broth dilution Minimum inhibitory concentration (MIC)
4. Beta lactamase enzyme detection4. Beta lactamase enzyme detection
Resistant Bacteria of ImportanceResistant Bacteria of Importance
MRSAMRSA methicillin resistant Staphylococcus aureusmethicillin resistant Staphylococcus aureus
VREVRE vancomycin resistant enterococcusvancomycin resistant enterococcus
ESBLESBL Extended Spectrum Beta Lactamase Gram neg rodsExtended Spectrum Beta Lactamase Gram neg rods
KPCKPC Klebsiella pneumonia Carbapenemase orKlebsiella pneumonia Carbapenemase or
CRECRE Carbapenamase Resistant EntericsCarbapenamase Resistant Enterics
Streptococcus pneumoniaeStreptococcus pneumoniae
Neisseria gonorrhoeaeNeisseria gonorrhoeae

8. Preparation of Bacteria forPreparation of Bacteria for
all Susceptibility Methodsall Susceptibility Methods
Pure culture of one organismPure culture of one organism
Log phase growth of bacteria - 16-24 hrs oldLog phase growth of bacteria - 16-24 hrs old
Methods test for stasis not killingMethods test for stasis not killing
Standardized suspension of bacteria:Standardized suspension of bacteria:
◦O.5 McFarland Standard – Barium sulfate solution thatO.5 McFarland Standard – Barium sulfate solution that
equals the turbidity of 10 8 bacteria/mlequals the turbidity of 10 8 bacteria/ml
◦Alternative method – use spectrophotometerAlternative method – use spectrophotometer
Incubation at 35 °C in room air (or CO²) for 18- 24 hrsIncubation at 35 °C in room air (or CO²) for 18- 24 hrs

9. 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

10. Quality ControlQuality Control
VerificationVerification: Before testing patients: Must correctly test multiple QC: Before testing patients: Must correctly test multiple QC
strains for 20 consecutive days or 3 replicates for 5 days. This is tostrains for 20 consecutive days or 3 replicates for 5 days. This is to
make sure you can perform the tests correctly.make sure you can perform the tests correctly.
ATCCATCC strains of bacteria (American Type Culture Collection) If QCstrains of bacteria (American Type Culture Collection) If QC
strains are within limits - you can then do Weekly quality control on allstrains are within limits - you can then do Weekly quality control on all
lots of cards, disks, plates in uselots of cards, disks, plates in use
◦ Data must be recorded and reviewed monthly by supervisorData must be recorded and reviewed monthly by supervisor
If weekly QC results are out of control:If weekly QC results are out of control:
◦ Immediately repeat/ inform supervisorImmediately repeat/ inform supervisor
◦ If repeat is OK – continue routine testingIf repeat is OK – continue routine testing
◦ If repeat is NOT OK – must investigate/document/ repeat 5 times toIf repeat is NOT OK – must investigate/document/ repeat 5 times to
start routine testing. All repeats must be in controlstart routine testing. All repeats must be in control

11. Agar Disk Diffusion (Kirby Bauer)Agar Disk Diffusion (Kirby Bauer)
Qualitative Susceptibility methodQualitative Susceptibility method
Mueller Hinton agar –with or without bloodMueller Hinton agar –with or without blood
◦150 mm plate diameter150 mm plate diameter
◦4mm in depth4mm in depth
◦Agar specifically balanced in Ca+ and Mg+,Agar specifically balanced in Ca+ and Mg+,
◦ if the ions are too high % amino-glycosides test falsely resistant,if the ions are too high % amino-glycosides test falsely resistant,
◦ if the ions too low % falsely susceptible amino-glycoside resultsif the ions too low % falsely susceptible amino-glycoside results
Streak bacteria on plate with cotton tipped swabStreak bacteria on plate with cotton tipped swab
Apply 6mm paper disks that contain single antibioticApply 6mm paper disks that contain single antibiotic
Incubate for 16-24 hrs at 35*CIncubate for 16-24 hrs at 35*C
Measure zone of diameter of inhibition of growth (mm)Measure zone of diameter of inhibition of growth (mm)

12. Kirby Bauer disk Dispenser
Each cartridge is a separate
antibiotic
Growth inside a zone is considered
resistance
Measure the diameter
of the zone of inhibition
Watch out for double
zones
Proteus will swarm
Into a zone

13. Kirby Bauer (KB)Kirby Bauer (KB)
Theory: Concentration gradient created with the diffusingTheory: Concentration gradient created with the diffusing
antibiotic and the increasing number of bacteria growing onantibiotic and the increasing number of bacteria growing on
the agar, this determines the zone size of inhibition aroundthe agar, this determines the zone size of inhibition around
disk.disk.
CLSI charts used to interpret the measured zone sizes asCLSI charts used to interpret the measured zone sizes as
Sensitive, Intermediate or ResistantSensitive, Intermediate or Resistant
Cannot directly compare zone sizes between antibiotics–Cannot directly compare zone sizes between antibiotics–
◦ex: ZID of 21mm zone size is as sensitive as a GM of 14mmex: ZID of 21mm zone size is as sensitive as a GM of 14mm
- zone sizes differ for organism/antibiotic combinations- zone sizes differ for organism/antibiotic combinations
◦Regression analysis can be used to calculate MIC valueRegression analysis can be used to calculate MIC value
related to KB zone sizerelated to KB zone size

14. E TestE Test
Quantitative MIC SusceptibilityQuantitative MIC Susceptibility
Calibrated plastic strips impregnated with one antibioticCalibrated plastic strips impregnated with one antibiotic
◦concentration gradient (mcg/ml) embedded in plasticconcentration gradient (mcg/ml) embedded in plastic
Diffusion gradient created as antibiotic diffuses intoDiffusion gradient created as antibiotic diffuses into
agar in an elliptical shapeagar in an elliptical shape
MIC (minimum inhibitory concentration) is where theMIC (minimum inhibitory concentration) is where the
ellipse ends on the plastic stripellipse ends on the plastic strip
Good method for slower growing fastidious organismsGood method for slower growing fastidious organisms
.

15. E Test method
E test method
Susceptibility result =
Where growth crosses
The plastic strip
E test for Strep pneumoniae
Low concentration
High concentration
MIC value
MIC value
Penicillin
Cefotaxime

16. Broth DilutionBroth Dilution
Quantitative Susceptibility MethodQuantitative Susceptibility Method
Bacteria inoculum: 0.5 McFarland standard – further diluted to 5x10Bacteria inoculum: 0.5 McFarland standard – further diluted to 5x1055
organisms/mlorganisms/ml
in brothin broth
Suspension is inoculated into tubes or micro titer trays containingSuspension is inoculated into tubes or micro titer trays containing
growth medium and known 2 fold dilutions (mcg/ml) of antibioticsgrowth medium and known 2 fold dilutions (mcg/ml) of antibiotics
Each horizontal row is a uniqueEach horizontal row is a unique
antibiotic – growth causesantibiotic – growth causes
turbidity in the wellsturbidity in the wells
Lowest dilution of antibioticLowest dilution of antibiotic
with No Growth (clear well)=with No Growth (clear well)=
MIC value.MIC value.

17. Broth Dilution DefinitionsBroth Dilution Definitions
MICMIC = lowest concentration of antibiotic inhibiting growth= lowest concentration of antibiotic inhibiting growth
MBCMBC = lowest concentration of antibiotic killing 99.9%= lowest concentration of antibiotic killing 99.9%
Antibiotic toleranceAntibiotic tolerance – Ratio of MBC/MIC (>=32)– Ratio of MBC/MIC (>=32)
◦MBC = 16 MIC= 8 16/8= 2MBC = 16 MIC= 8 16/8= 2 No ToleranceNo Tolerance
◦MBC = 128 MIC = 8MBC = 128 MIC = 8 128/2 = 64128/2 = 64 Tolerance*Tolerance*

18. Siemens Microscan walkaway
AST system
BD Phoenix AST system
Biomerieux Vitek2 AST
Automated Identification
Susceptibility Testing
Systems (AST)

19. Disk test for Beta lactamaseDisk test for Beta lactamase
Detection (Cefinase Test)Detection (Cefinase Test)
Add bacteria to filter paper impregnated with NitrocefinAdd bacteria to filter paper impregnated with Nitrocefin
(yellow colored/chromogenic cephalosporin substrate)(yellow colored/chromogenic cephalosporin substrate)
Incubate at room temp (@ 1 minute) and observeIncubate at room temp (@ 1 minute) and observe
for color change from yellow to redfor color change from yellow to red
Beta lactamase enzyme of bacteria breaks down the betaBeta lactamase enzyme of bacteria breaks down the beta
lactam ring of Nitrocefin to produce a red end productlactam ring of Nitrocefin to produce a red end product
Detects resistance to Ampicillin/Penicillin/1Detects resistance to Ampicillin/Penicillin/1stst
gen Cephalosporingen Cephalosporin
in Haemophilus, N. gonorrhoea , Moraxella catarrhalis,in Haemophilus, N. gonorrhoea , Moraxella catarrhalis,
Enterococcus, and anaerobic gram negative rodsEnterococcus, and anaerobic gram negative rods
This test does NOT detect Extended Spectrum Beta LactamaseThis test does NOT detect Extended Spectrum Beta Lactamase
enzyme produced by enteric gram negative rodsenzyme produced by enteric gram negative rods

20. Beta lactamase detectionBeta lactamase detection
tidbitstidbits
Haemophilus influenzaeHaemophilus influenzae
◦In US, approx 28% are beta lactamase producers andIn US, approx 28% are beta lactamase producers and
therefore, resistant to Ampicillintherefore, resistant to Ampicillin
Bacteroides fragilis groupBacteroides fragilis group
◦Primary resistance mechanism is beta lactamase productionPrimary resistance mechanism is beta lactamase production
>95% of strains are resistant to Pencillin>95% of strains are resistant to Pencillin
Moraxella catarrhalisMoraxella catarrhalis
◦>90% beta lactamase positive /Ampicillin resistant>90% beta lactamase positive /Ampicillin resistant

21. Methicillin Resistant StaphMethicillin Resistant Staph
aureus (MRSA)aureus (MRSA)
Test for oxacillin (OX) resistance - it is more stable for testingTest for oxacillin (OX) resistance - it is more stable for testing
than methicillin in the laboratorythan methicillin in the laboratory
OLDOLD - If- If OX is resistantOX is resistant - S. aureus is reported as a MRSA- S. aureus is reported as a MRSA
NEW - resistanceNEW - resistance testing for Cefoxitin is more reliable and atesting for Cefoxitin is more reliable and a
preferred way to confirm MRSApreferred way to confirm MRSA
All cephalosporin antibiotics are reported resistant for MRSAAll cephalosporin antibiotics are reported resistant for MRSA
and should never be used for therapyand should never be used for therapy
Resistance mechanism byResistance mechanism by penicillin binding proteins (PBPs)penicillin binding proteins (PBPs)
◦ PBPs bind penicillin and related antibioticsPBPs bind penicillin and related antibiotics
◦ The binding prevents disruption of the peptidoglycan synthesis in theThe binding prevents disruption of the peptidoglycan synthesis in the
Staph aureus cell wallStaph aureus cell wall
◦ PBPs are produced by thePBPs are produced by the mecA genemecA gene

22. Oxacillin KB disk = resistant
MRSA
Cefoxitin KB disk
Newer method –
more sensitive
screen for MRSA
Cefoxitin (FOX)
KB = sensitive
S. aureus
Methods to Detect Methicillin and
Oxacillin Resistance - MRSA
Oxacillin Testing is no longer
the preferred method for
detection

23. Clindamycin Induction Test –Clindamycin Induction Test –
The D testThe D test
This test determines if Staph aureus, including MRSA, isThis test determines if Staph aureus, including MRSA, is
susceptible to Clindamycin more reliably than testingsusceptible to Clindamycin more reliably than testing
Clindamycin resistance by KB or MICClindamycin resistance by KB or MIC
During antibiotic therapy, S aureus isolates resistant toDuring antibiotic therapy, S aureus isolates resistant to
Erythromycin possess enzymes that can be induced toErythromycin possess enzymes that can be induced to
make the S. aureus also resistant to Clindamycinmake the S. aureus also resistant to Clindamycin
Clindamycin is often used to treat serious soft tissueClindamycin is often used to treat serious soft tissue
infections with MRSA – so reliable testing necessaryinfections with MRSA – so reliable testing necessary

24. D test Negative-
round Clindamycin zone
Kirby Bauer zone around Clindamycin will beKirby Bauer zone around Clindamycin will be
blunted to form a D if Clindamycin can beblunted to form a D if Clindamycin can be
induced by Erythromycin to be resistant – soinduced by Erythromycin to be resistant – so
called INDUCIBLE RESISTANCE.called INDUCIBLE RESISTANCE.
Clindamycin should be reported as resistant byClindamycin should be reported as resistant by
clindamycin induction and not used for therapyclindamycin induction and not used for therapy
The D Test

25. EnterococcusEnterococcus
All are intrinsically resistant to:All are intrinsically resistant to:
◦CephalosporinsCephalosporins
◦ClindamycinClindamycin
◦Trimethoprim/sulfamethoxazoleTrimethoprim/sulfamethoxazole
Synergistic antibiotic therapy can be important for theSynergistic antibiotic therapy can be important for the
treatment of Enterococcustreatment of Enterococcus
◦Ampicillin plus Gentamicin is synergistic = increasedAmpicillin plus Gentamicin is synergistic = increased
killing potential in combinationkilling potential in combination
◦Important for endocarditis therapyImportant for endocarditis therapy

26. Vancomycin Resistant EnterococcusVancomycin Resistant Enterococcus
(VRE)(VRE)
Acquired resistance to vancomycin –Acquired resistance to vancomycin –
◦Plasmid mediated vanA associated with E. faeciumPlasmid mediated vanA associated with E. faecium
◦Plasmic mediated vanB associated with E. faecalisPlasmic mediated vanB associated with E. faecalis
Detected by KB, automated AST systems, and EtestDetected by KB, automated AST systems, and Etest
methodsmethods
Drugs of choice limited if VRE detected –Drugs of choice limited if VRE detected –
◦ LinezolidLinezolid
◦ Synercid for E. faecium onlySynercid for E. faecium only
Major infection control issue!!Major infection control issue!!
◦Rectal colonization can contaminatie the environment andRectal colonization can contaminatie the environment and
lead to transmission to surrounding patientslead to transmission to surrounding patients
◦Most infections related to ICU stays and longMost infections related to ICU stays and long
hospitalizationshospitalizations
◦Not virulent but problematic in immune suppressedNot virulent but problematic in immune suppressed

27. Extended Spectrum BetaExtended Spectrum Beta
LactamaseLactamase
[ESBL][ESBL]
Enzymes produced by Enteric Gram negative bacilliEnzymes produced by Enteric Gram negative bacilli
◦Confer resistance to Cephalosporins, Penicillins andConfer resistance to Cephalosporins, Penicillins and
Monobactam (Aztreonam) by opening the beta lactam ringMonobactam (Aztreonam) by opening the beta lactam ring
and inactivating the antibioticand inactivating the antibiotic
◦ESBLs do not attack Cephamycin (cefoxitin, cefotetan) or theESBLs do not attack Cephamycin (cefoxitin, cefotetan) or the
Carbapenem antibiotic classesCarbapenem antibiotic classes
Plasmid mediated CTX-M beta lactamases are the mostPlasmid mediated CTX-M beta lactamases are the most
common in the US currently, but many more ESBL typescommon in the US currently, but many more ESBL types
worldwideworldwide
Therapy for ESBL producing gram negative rods:Therapy for ESBL producing gram negative rods:
◦Carbapenems: Imipenem, Meropenem, Doripenem,Carbapenems: Imipenem, Meropenem, Doripenem,
ErtapenemErtapenem

28. ESBL Susceptibility PatternESBL Susceptibility Pattern
Escherichia coli – ESBL CTX-M PositiveEscherichia coli – ESBL CTX-M Positive
◦ AmpicillinAmpicillin RR
◦ CefazolinCefazolin RR
◦ GentamicinGentamicin RR
◦ CefotetanCefotetan SS (Cephamycins are not cephalosporins)(Cephamycins are not cephalosporins)
◦ CefotaximeCefotaxime RR
◦ CeftazidimeCeftazidime RR
◦ CefpodoximeCefpodoxime RR
◦ PiperacillinPiperacillin RR
◦ Pip/Tazobactam SPip/Tazobactam S /R (?)/R (?) Tazobactam is a beta lactam blockerTazobactam is a beta lactam blocker
◦ ImipenemImipenem SS **(Carbapenem) – Antibiotic of choice**(Carbapenem) – Antibiotic of choice

29. Detecting ESBL in the
laboratory
Standard of Practice:Standard of Practice:
◦CLSI established new Cephalosporin MIC and KBCLSI established new Cephalosporin MIC and KB
breakpoints to safely detect ESBL activitybreakpoints to safely detect ESBL activity
◦New MIC breakpoints are one to three doubling dilutionsNew MIC breakpoints are one to three doubling dilutions
lower than previously used andlower than previously used and
◦New KB zones for susceptible are largerNew KB zones for susceptible are larger
◦These values were adjusted to aid laboratories in theThese values were adjusted to aid laboratories in the
detection of ESBLdetection of ESBL
Molecular testing needed for confirmation of actual enzymeMolecular testing needed for confirmation of actual enzyme
present (CTX-M)– beyond the scope of most clinicalpresent (CTX-M)– beyond the scope of most clinical
laboratorieslaboratories

30. Positive ESBL Double Disk TestPositive ESBL Double Disk Test
*Older method for ESBL Detection*Older method for ESBL Detection –– observeobserve
action of the Beta lactam enzyme blockeraction of the Beta lactam enzyme blocker
Resistant
Cefotaxime
Resistant
Ceftazidime
Susceptible
Ceftazidime plus
Clavulanic acid (beta
lactam blocker)
Susceptible
Cefotaxime plus
Clavulanic acid (beta
Lactam blocker

31. Why all the fuss about
ESBLs?
GNRs with ESBL phenotypes >=10% in US and the numbers are
increasing
Limited treatment options
◦ Carbapenems: meropenem, imipenem, ertapenem
Risk factors:
◦ Long hospital stay – particularly in the ICU
◦ Central lines
◦ Issues with the intestine
◦ Long term care facility
◦ Ventilator assistance

32. Carbapenemases - CRE
Carbapenem antibiotics currently have the highest spectrum of
activity against multi-drug resistant GNRs
But the worst scenario has come true – Appearance of
Carbapenem-hydrolyzing-beta-lactamases which confer
resistance to a broad spectrum of beta lactam antibiotics –
making GNR pan resistant
CRE – Carbapenemase Resistant Enteriobacteriaceae
Two CREs are getting the most attention:
◦ KPC – “Klebsiella pneumoniae carbapenemas most common in the US
◦ NDM-1 – New Delhi metallo-beta-lactamase has received much press.
Recognized in 2009. Resistance determinants are numerous and great
concern about its spread.
Infections with CRE producing GNRs - 50% fatality rate

33. Most sensitive screen to detected CRE is an
Ertapenem MIC test. However, all
Carbapenem antibiotics will show elevated
MICs in the resistance range.
Treatment of CRE:
Polymyxins (Colistin and Polymyxin B)
These antibiotics are +/- toxic and
problematic for therapy.
Carbapenemase Resistant Enterics (CRE)
Laboratory Testing

34. OLD Method to Detect CRE/KPC/NDM-1OLD Method to Detect CRE/KPC/NDM-1
Modified Hodge TestModified Hodge Test
Observe growth on this plate:
Background is lawn of E. coli.
Patient GNR streaks hug the
Imipenem disc in the center of the
plate. (A, B, C , D and E)
A = Hodge Test Positive
Therefore, it is a CRE!
Shoulder effect around streak of
test organism (arrow)
Negative = “B”
No shoulder effect, this organism
is not a CRE
Lawn of E. coli
Streaks of patient
GNR

35. Streptococcus pneumoniae andStreptococcus pneumoniae and
resistance to Penicillin (PEN)resistance to Penicillin (PEN)
Two step resistance test – old wayTwo step resistance test – old way
◦Step 1Step 1 -- Oxacillin KBOxacillin KB disk testing performeddisk testing performed
◦If resistant to Oxacillin, possible resistance to PENIf resistant to Oxacillin, possible resistance to PEN
◦Step 2Step 2 –Confirm PEN resistance by MIC test–Confirm PEN resistance by MIC test
◦MIC value determines susceptibility or resistanceMIC value determines susceptibility or resistance
Best method – Primary test is to perform a Penicillin MICBest method – Primary test is to perform a Penicillin MIC
test by E Test or broth dilutiontest by E Test or broth dilution
KB method cannot be used to test PEN against StrepKB method cannot be used to test PEN against Strep
pneumoniae, under predicts resistancepneumoniae, under predicts resistance

36. Strep pneumoniae MICStrep pneumoniae MIC
valuesvalues
CLSI interpretation of AST dependent on the site of infectionCLSI interpretation of AST dependent on the site of infection
◦CSFCSF Blood/RespBlood/Resp
◦Sensitive <=0.06 mcg/mlSensitive <=0.06 mcg/ml <=2 mcg/ml<=2 mcg/ml
◦Intermediate 0.12 - 1 mcg/mlIntermediate 0.12 - 1 mcg/ml <=4<=4
◦ResistantResistant >= 2 mcg/ml>= 2 mcg/ml >=8>=8
High level PEN resistance is <=10% in USHigh level PEN resistance is <=10% in US
◦If Pen resistant- antibiotics of choice become 3If Pen resistant- antibiotics of choice become 3rdrd
gengen
Cephalosporin, vancomycin or quinoloneCephalosporin, vancomycin or quinolone

37. Neisseria gonorrhoeae
(GC)
Increasing resistance of GC over last two decades
◦1980’s beta lactamase producing - Penicillin resistance
◦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 (PBPs) 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 that only
test for GC and not for resistance markers