Susceptibility Testing Review
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Antibiotic Sensitivity Testing

Antibiotic Sensitivity Testing

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Susceptibility Testing Review Presentation Transcript

  • 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