Antibiotic Sensitivity Testing

1. Antibiotic SensitivityAntibiotic Sensitivity
TestingTesting
Margie A. Morgan, PhD MT(ASCP),Margie A. Morgan, PhD MT(ASCP),
D(ABMM)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

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 mechanisms ofThe Four Major mechanisms of
antibiotic resistanceantibiotic resistance
 Enzymatic cleavage leads to inactivation of antibioticEnzymatic cleavage leads to inactivation of antibiotic
 Beta lactam and aminoglycoside antibioticsBeta lactam and aminoglycoside antibiotics
 Active Beta lactamases and amino-glycoside modifying enzymesActive Beta lactamases and amino-glycoside modifying enzymes
 Altered receptors/binding proteins preventing attachment ofAltered receptors/binding proteins preventing attachment of
antibiotics to the bacterial surfaceantibiotics to the bacterial surface
 Altered Penicillin binding proteinsAltered Penicillin binding proteins
 Strep pneumonia resistance to penicillinStrep pneumonia resistance to penicillin
 MRSA resistance to methicillinMRSA resistance to methicillin
 Altered permeability/influx and efflux pumps stopping passageAltered permeability/influx and efflux pumps stopping passage
through porins – gram negative bacillithrough porins – gram negative bacilli
 Pseudomonas resistance to amino-glycosidesPseudomonas resistance to amino-glycosides
 Bypass of a metabolic block/metabolic block imposed byBypass of a metabolic block/metabolic block imposed by
antibioticantibiotic
 Enterococcus resistance to TMP/SXTEnterococcus resistance to TMP/SXT

6. The Rules for SusceptibilityThe Rules for Susceptibility
TestingTesting
1. CLSI – Clinical Laboratory Standards Institute1. CLSI – Clinical Laboratory Standards Institute
Approved standards for the testing & reportingApproved standards for the testing & reporting
of susceptibility results/ updated yearlyof 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
2. Susceptibility Tests are tests of bacterial stasis –2. Susceptibility Tests are tests of bacterial stasis –
not killingnot killing

7. Methods/Bacteria in ReviewMethods/Bacteria in Review
METHODSMETHODS
1. Kirby Bauer disk diffusion1. Kirby Bauer disk diffusion
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 IN THE NEWSRESISTANT BACTERIA IN THE NEWS
 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 or CREKlebsiella pneumonia Carbapenemase or CRE
(Carbapenamase Resistant Enterics)(Carbapenamase Resistant Enterics)
 Streptococcus pneumoniaStreptococcus pneumonia
 Neisseria gonorrhoeaeNeisseria gonorrhoeae

8. Preparation of Bacteria for allPreparation of Bacteria for all
Susceptibility MethodsSusceptibility Methods
Requires pure culture of one organism onlyRequires pure culture of one organism only
 Log phase growth of bacteria - 16-24 hrs oldLog phase growth of bacteria - 16-24 hrs old
 Standardized suspension of bacteria must beStandardized suspension of bacteria must be
prepared using:prepared using:
 O.5 McFarland Standard – Barium sulfate solutionO.5 McFarland Standard – Barium sulfate solution
that equals the turbidity of @ 10 8 bacteria/mlthat equals the turbidity of @ 10 8 bacteria/ml
 Alternative method – use spectrophotometerAlternative method – use spectrophotometer
 Incubation of tests are at 35 *C in room airIncubation of tests are at 35 *C in room air
(some require CO2) for 18- 24 hrs(some require 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 Control for all methodsQuality Control for all methods
Before you can test patients: Must test multiple QC strains for 20Before you can test patients: Must test multiple QC strains for 20
consecutive days or 3 replicates for 5 days plan. This is to makeconsecutive days or 3 replicates for 5 days plan. This is to make
sure you can perform the tests correctly.sure you can perform the tests correctly.
The QC values must be within CLSI established limits usingThe QC values must be within CLSI established limits using
ATCC strains of organisms (American Type Culture Collection)ATCC strains of organisms (American Type Culture Collection)
If QC strains are within limits - you can then do Weekly qualityIf QC strains are within limits - you can then do Weekly quality
control on all lots of cards, disks, plates in usecontrol on all lots 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 5If repeat is NOT OK – must investigate/document/ repeat 5
times to start routine testing. All repeats must be in controltimes to start routine testing. All repeats must be in control

11. Agar Disk Diffusion (Kirby BauerAgar Disk Diffusion (Kirby Bauer
Method)Method)
Procedure: Qualitative Susceptibility methodProcedure: Qualitative 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 contains single antibioticApply 6mm paper disks that contains 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

13. Kirby Bauer
Disk Diffusion
Measure diameter in mm– across center of the
Disk – for each antibiotic disk
Colonies growing into zones is considered
Resistant to that antibiotic
Growth inside a zone is considered
resistance
Measure diameter of the
Zone of inhibition in mm

14. Double zoning - measure the inside zone
Watch out! The sensi could
Be mixed – NEVER read a mixed sensi!
Proteus swarms into zone and is not
considered as resistant

15. Kirby Bauer (KB)Kirby Bauer (KB)
 Concentration gradient created with the diffusingConcentration gradient created with the diffusing
antibiotic and the increasing number of bacteriaantibiotic and the increasing number of bacteria
growing on the agar, this determines the zone ofgrowing on the agar, this determines the zone of
inhibition around disk.inhibition around disk.
 CLSI charts used to interpret the measured zone sizesCLSI charts used to interpret the measured zone sizes
as Sensitive, Intermediate or Resistantas Sensitive, Intermediate or Resistant
 Cannot directly compare zone sizes betweenCannot directly compare zone sizes between
antibiotics–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 value relatedRegression analysis can be used to calculate MIC value related
to KB zone sizeto KB zone size

16. E TestE Test
Quantitative MIC SusceptibilityQuantitative MIC Susceptibility
 Calibrated plastic strips impregnated with oneCalibrated plastic strips impregnated with one
antibiotic/concentration gradient (mcg/ml) embeddedantibiotic/concentration gradient (mcg/ml) embedded
in plastic / carefully placed on the agar surfacein plastic / carefully placed on the agar surface
 Gradient created as antibiotic diffuses into agar in anGradient created as antibiotic diffuses into agar in an
elliptical shapeelliptical 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
 Useful for any organism but a method of choice forUseful for any organism but a method of choice for
slow growing fastidious organismsslow growing fastidious organisms

17. E Test method
E test method
Susceptibility result =
Where growth crosses
The plastic strip
E test for Strep pneumonia susceptibility
Low concentration
High concentration
MIC value
MIC value

18. Broth DilutionBroth Dilution
Quantitative Susceptibility MethodQuantitative Susceptibility Method
 Bacteria inoculum: 0.5 McFarland standard –Bacteria inoculum: 0.5 McFarland standard –
further diluted to 5x10further diluted to 5x105organisms/ml5organisms/ml
in brothin broth
 Suspension is inoculated into tubes or microSuspension is inoculated into tubes or micro
titer trays containing growth medium andtiter trays containing growth medium and
known 2 fold dilutions (mcg/ml) of antibioticsknown 2 fold dilutions (mcg/ml) of antibiotics

19. Doubling dilution MIC Tray
Each row has one antibiotic in >= 4 dilutions

20. Broth Dilution DefinitionsBroth Dilution Definitions
 MICMIC = lowest concentration of antibiotic inhibiting growth= lowest concentration of antibiotic inhibiting growth
 2 fold dilutions:2 fold dilutions: 1 2 41 2 4 88 16 32 64 128 mcg/ml16 32 64 128 mcg/ml
 GrowthGrowth No growthNo growth
 MIC = 8 mcg/mlMIC = 8 mcg/ml
 MBCMBC - Minimum- Minimum bactericidalbactericidal concentration determined by theconcentration determined by the
subculture of the contents of the wells that show no growth tosubculture of the contents of the wells that show no growth to
solid agar - the lowest concentration of antibiotic that kills 99.9%solid agar - the lowest concentration of antibiotic that kills 99.9%
of original inoculum is the MBC.of original inoculum is the MBC.
 8 168 16 3232 64 12864 128 32mcg/ml = MBC32mcg/ml = MBC
 GrowthGrowth No GrowthNo Growth
 Antibiotic toleranceAntibiotic tolerance - MBC/MIC ratio >=32- MBC/MIC ratio >=32
 MBC = 128 MIC= 2 128/2= 64MBC = 128 MIC= 2 128/2= 64 ToleranceTolerance

21. Siemens Microscan walkaway
AST system
BD Phoenix AST system
Biomerieux Vitek2 AST

22. 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)(yellow colored/chromogenic cephalosporin)
 Incubate at room temp (@ 1 minute) and observe for colorIncubate at room temp (@ 1 minute) and observe for color
change from yellow to redchange from yellow to red
 Positive result is color changePositive result is color change to redto red ––
 Bacteria beta lactamase enzyme breaks down the beta lactam ring of Nitocefin toBacteria beta lactamase enzyme breaks down the beta lactam ring of Nitocefin to
produce a red end productproduce a red end product
 Detects resistance to Ampicillin/Penicillin/Cephalosporins inDetects resistance to Ampicillin/Penicillin/Cephalosporins in
Haemophilus, Neisseria gonorrhoea , Moraxella catarrhalis,Haemophilus, Neisseria 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

23. Beta lactamase detection tidbitsBeta lactamase detection tidbits
 Haemophilus influenzaHaemophilus influenza
 In US, approx 28% are beta lactamase producersIn US, approx 28% are beta lactamase producers
therefore, resistant to Ampicillintherefore, resistant to Ampicillin
 Bacteroides fragilis groupBacteroides fragilis group
 Primary resistance mechanism is beta lactamasePrimary resistance mechanism is beta lactamase
production –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

24. Methicillin Resistant StaphMethicillin Resistant Staph
aureus (MRSA)aureus (MRSA)
 In the laboratory you test oxacillin (OX) because it is more stableIn the laboratory you test oxacillin (OX) because it is more stable
for testing than methicillinfor testing than methicillin
 IfIf OX is resistantOX is resistant the S. aureus is reported as a MRSAthe S. aureus is reported as a MRSA
 Cefoxitin resistanceCefoxitin resistance testing is also a very reliable and atesting is also a very reliable and a
preferred way to confirm a S. aureus to be a MRSApreferred way to confirm a S. aureus to be a MRSA
 All cephalosporin antibiotics are reported as resistant whenAll cephalosporin antibiotics are reported as resistant when
reporting results for MRSA and should never be used for therapyreporting results for MRSA and should never be used for therapy
 Resistance mechanism is byResistance mechanism is by penicillin binding proteins (PBP)penicillin binding proteins (PBP)
 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
 PBP are produced by thePBP are produced by the mecA genemecA gene

25. Oxacillin KB= resistant
Newer and more
sensitive
method to
screen for MRSA
is using a
Cefoxitin KB to
determine
Methicillin
resistance
Methods to Detect Methicillin and
Oxacillin Resistance
Oxacillin is no longer
suggested by CLSI for
detecting MRSA

26. Clindamycin Induction TestClindamycin Induction Test
 Also known as the D testAlso known as the D test
 This test accurately determines if Staph aureus,This test accurately determines if Staph aureus,
including MRSA, is susceptible to Clindamycinincluding MRSA, is susceptible to Clindamycin
 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
 Kirby Bauer zone around Clindamycin will be bluntedKirby Bauer zone around Clindamycin will be blunted
to form a D if Clindamycin can be induced byto form a D if Clindamycin can be induced by
Erythromycin to be resistant – so called INDUCIBLEErythromycin to be resistant – so called INDUCIBLE
RESISTANCE. Clindamycin should be reported asRESISTANCE. Clindamycin should be reported as
resistant by clindamycin induction and not used forresistant by clindamycin induction and not used for
therapy. Clinda is often used to treat skin infections.therapy. Clinda is often used to treat skin infections.

27. D test Positive
Blunt D shaped CC zone
Clindamycin has inducible
resistance
D test Negative
Round CC zone

28. EnterococcusEnterococcus
 All naturally resistant to:All naturally resistant to:
 CephalosporinsCephalosporins
 ClindamycinClindamycin
 Trimethoprim/sulfamethoxazoleTrimethoprim/sulfamethoxazole
 Synergistic antibiotic therapy can be important for the treatmentSynergistic antibiotic therapy can be important for the treatment
of Enterococcusof Enterococcus
 Ampicillin plus Gentamicin is synergistic and often used toAmpicillin plus Gentamicin is synergistic and often used to
increase the killing potentialincrease the killing potential
 Important for endocarditis therapyImportant for endocarditis therapy

29. Vancomycin Resistant EnterococcusVancomycin Resistant Enterococcus
(VRE)(VRE)
 Acquired resistance to vancomycin –Acquired resistance to vancomycin –
 Plasmid mediated vanA associtated with E. faeciumPlasmid mediated vanA associtated with E. faecium
 Plasmic mediated vanB associated with E. faecalisPlasmic mediated vanB associated with E. faecalis
 Not difficult to detect – vancomycin resistance easily detected byNot difficult to detect – vancomycin resistance easily detected by
KB, automated AST systems, and Etest methodsKB, automated AST systems, and Etest methods
 Drugs of choice if VRE detected –Drugs of choice if VRE detected –
 LinezolidLinezolid
 Synercid for E. faecium onlySynercid for E. faecium only
 Major epidemiology issues!!Major epidemiology issues!!
 Rectal colonization can contaminatie the environment andRectal colonization can contaminatie the environment and
lead to transmission to patientslead to transmission to patients
 Most infections related to ICU stays and long hospitalizationsMost infections related to ICU stays and long hospitalizations
 Not virulent but problematic in immune suppressedNot virulent but problematic in immune suppressed

30. Extended Spectrum Beta LactamaseExtended Spectrum Beta Lactamase
[ESBL][ESBL]
 Enzymes that are produced by Gram negative bacteriaEnzymes that are produced by Gram negative bacteria
 Confer resistance to Cephalosporins, Penicillins and MonobactamConfer resistance to Cephalosporins, Penicillins and Monobactam
(Aztreonam) by opening the beta lactam ring inactivating the antibiotic(Aztreonam) by opening the beta lactam ring inactivating the antibiotic
 Cannot attack cephamycins (cefoxitin, cefotetan) or the carbapenemsCannot attack cephamycins (cefoxitin, cefotetan) or the carbapenems
(imipenem, meropenem, ertapenem, doripenem)(imipenem, meropenem, ertapenem, doripenem)
 Generally susceptible to beta-lactamase inhibitors (tazobactam)Generally susceptible to beta-lactamase inhibitors (tazobactam)
 Plasmid mediated TEM, SHV, CTX-M beta lactamases are thePlasmid mediated TEM, SHV, CTX-M beta lactamases are the
most commonmost common
 Therapy for ESBL producing gram negative rods:Therapy for ESBL producing gram negative rods:
 Carbapenems: Imipenem, Meropenem, Doripenem, ErtapenemCarbapenems: Imipenem, Meropenem, Doripenem, Ertapenem
 Piperacillin/Tazobactam – Tazobactam blocks beta lactamase actionPiperacillin/Tazobactam – Tazobactam blocks beta lactamase action

31. ESBL Susceptibility PatternESBL Susceptibility Pattern
 Escherichia coli – ESBL PositiveEscherichia coli – ESBL 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 (Tazobactam is a beta lactam blocker)(Tazobactam is a beta lactam blocker)
 ImipenemImipenem SS (Carbapenem)(Carbapenem)

32. Detecting ESBL in the laboratory
 Suggested method:Suggested method:
 Cephalosporin MIC values and KB zone sizesCephalosporin MIC values and KB zone sizes
established by CLSI to safely detect ESBL activityestablished by CLSI to safely detect ESBL activity
 New breakpoints are one to three doubling dilutionsNew breakpoints are one to three doubling dilutions
lower than breakpoints and KB zones forlower than breakpoints and KB zones for
susceptible that were used for decadessusceptible that were used for decades
 These values were lowered to aid laboratories in theThese values were lowered to aid laboratories in the
correct detection of ESBLcorrect detection of ESBL
 Molecular testing needed for confirmation –Molecular testing needed for confirmation –
beyond the scope of most clinical laboratoriesbeyond the scope of most clinical laboratories

33. 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

34. Positive ESBL Double Disk TestPositive ESBL Double Disk Test
Resistant
Cefotaxime
Resistant
Ceftazidime
Susceptible
Ceftazidime plus
Clavulinic acid
Susceptible
Cefotaxime plus
Clavulinic acid

35. 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

36. 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.

37. 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%

38. Most 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 and problematic for
therapy.
Carbapenemase Resistant Enterics (CRE)
Laboratory Testing

39. 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
Test GNR streaks hug the
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
Lawn of E. coli
Streaks of test
GNR

40. Streptococcus pneumonia andStreptococcus pneumonia and
resistance to Penicillin (PEN)resistance to Penicillin (PEN)
 Two step resistance testing can be usedTwo step resistance testing can be used
 Step 1Step 1 -- Oxacillin KBOxacillin KB disk testingdisk testing
 If resistant to Oxacillin possible resistance to PENIf resistant to Oxacillin possible resistance to PEN
 Step 2Step 2 -Must confirm PEN resistance by MIC test-Must confirm PEN resistance by MIC test
methodmethod
 MIC value determines susceptibility or resistanceMIC value determines susceptibility or resistance
 Best most direct method one step- Perform aBest most direct method one step- Perform a
Penicillin MIC test by E Test or broth dilutionPenicillin MIC test by E Test or broth dilution
[cannot use KB to test Penicillin][cannot use KB to test Penicillin]

41. Strep pneumonia MIC valuesStrep pneumonia MIC values
 Penicillin MIC values – CLSI has setPenicillin MIC values – CLSI has set
interpretation depending on site of infectioninterpretation depending on 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 uncommon,High level PEN resistance is uncommon,
usually <= 10%usually <= 10%
 If resistant to Penicillin antibiotics of choiceIf resistant to Penicillin antibiotics of choice
become Cefotaxime, vancomycin or a quinolonebecome Cefotaxime, vancomycin or a quinolone

42. Neisseria gonorrhoeae (GC)
 Increasing resistance of GC over last two decades
 1980’s 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