ESBL - Dynamics and Detection

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ESBL - Dynamics and Detection

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ESBL - Dynamics and Detection

  1. 1. ESBLEXTENDED SPECTRUM BETA LACTAMASES DYNAMICS AND DETECTION DR.T.V.RAO 1Dr.T.V.Rao MD
  2. 2. SURVIVAL OF THE FITTEST Resistant bacteria survive, susceptible ones die Mutant emerges Sensitive cells Mutant’s progeny2 slowly killed by antibiotic overrun
  3. 3. PENICILLIN BETA LACTAM RING BETA LACTAMASES enzymes that inactivate the beta-lactam ringCEPHALOSPORIN BETA LACTAM RING 3 Dr.T.V.Rao MD
  4. 4. ACTION OF A B-LACTAMASE S N H2O Inactive penicilloateO COOH SActive penicillin HN O OH COOH 4Dr.T.V.Rao MD
  5. 5. BASIS OF BETALACTAMSE ACTIVITYTime-delayed Growth. Beta-lactamase (red) isproduced by the centralcolony, promoting growthof nearby, non-resistantcolonies as it deactivatesampicillin (blue). Diffusionof beta lactamase throughagar leads to time-delayedgrowth of non-resistantcolonies 5 Dr.T.V.Rao MD
  6. 6. B-LACTAM ANTIBIOTICSPenicillins •Ampicillin •PiperacillinBeta-lactam/beta-lactamase inhibitors •Ampicillin/sulbactam •Amoxicillin/clavulanate •Ticarcillin/clavulanate •Piperacillin/Tazobactam 6 Dr.T.V.Rao MD
  7. 7. PENICILLINS1st gen – strep infection (G+) Ex. Penicillin,CloxacillinExtended-spectrum – have broaderspectrum against G- including E.Coli Ex.Amoxicillin With inhibitor would protectagainst some beta-lactamase producers Ex.Amoxicillin/ClavBroad spectrum – manyEnterobacteriaceae ExPiperacillin/Tazobactam 7Dr.T.V.Rao MD
  8. 8. Some beta-lactamases only inactivate a small number of antibiotics e.g. penicillin extended Others have spectrum to all the penicillins and cephalosporins e.g. cefuroxime, ceftriaxone (ESBLs) In addition may also carry resistance to other antibiotics e.g. ciprofloxacin. 8Dr.T.V.Rao MD
  9. 9. DEFINITION OF ESBL : Class A by Ambler or Group 2be by Bush classifications Typically, enzymes are plasmid- mediated derived from older ß- lactamases of TEM and SHV In early 2000s, CTX-M derived ß- lactamases are included 9Dr.T.V.Rao MD
  10. 10. ESBL EVOLUTIONMid 1980sVariants of TEM and SHVBreakdown 3rd generationcephalosporinsMainly in hospital KlebsiellaSpread world wide 10Dr.T.V.Rao MD
  11. 11. WHAT ARE EXTENDED-SPECTRUM Β-LACTAMASES?ESBLs are enzymes that mediate resistanceto extended-spectrum (third generation)cephalosporins (e.g., ceftazidime,cefotaxime, and ceftriaxone) andmonobactams (e.g., aztreonam) but do notaffect cephamycins (e.g., cefoxitin andCefotetan) or carbapenems (e.g.,meropenem or imipenem). 11 Dr.T.V.Rao MD
  12. 12. AMBLER CLASSIFICATION OF Β- LACTAMASES β-lactamases Active site Serine-enzymes Zinc-enzymes Nucleotide sequence A C D B Four evolutionarily distinct molecular classesDr.T.V.Rao MD
  13. 13. WHAT IS A BETA-LACTAM? Abx • Penicillin • Cephalosporin • Monobactam • Carbapenem Bacteriocidal Google Images 13Dr.T.V.Rao MD
  14. 14. CEPHALOSPORINS 4th 3rd 2nd 1st 14 Willey, et al., 2008Dr.T.V.Rao MD
  15. 15. CEPHALOSPORINS-USES1st gen: strep, staph, G- including E.coli Ex.Cefazolin2nd gen: greater spectrum against G- Ex.Cefoxitin3rd gen: even greater activity, combatnarrow-spectrum beta-lactamase producers ESBLs emerged Ex. Ceftazidime4th gen: effective against G- bacilliexpressing Xm AmpC resistant to 3rd genEx. Cefepime 15Dr.T.V.Rao MD
  16. 16. OTHERSMonobactamsMonobactams very active against G-including E.coli Ex. AztreonamCarbapenemsCarbapenems have an extremelybroad spectrum. Cross-reactivity withpenicillins or cephalosporins Ex.Imipenam 16Dr.T.V.Rao MD
  17. 17. THE FIGHT GOES ON ..Beta-lactamBeta-lactamaseBeta-lactamase inhibitor Google ImagesESBL 17Dr.T.V.Rao MD
  18. 18. COMMON ESBL PRODUCERS Klebsiella pneumoniae Escherichia coli Proteus mirabilis Enterobacter cloacae Non-typhoidal Salmonella (in some countries) 18Dr.T.V.Rao MD
  19. 19. THE FIGHT BETA-LACTAM PG N cell O 19Dr.T.V.Rao MD LYSIS
  20. 20. THE FIGHT BETA-LACTAMASE PG beta-lactamase N cell O 20Dr.T.V.Rao MD
  21. 21. THE FIGHT BETA-LACTAMASE PG O NH OH cell 21Dr.T.V.Rao MD
  22. 22. THE FIGHT BETA-LACTAMASE INHIBITOR PG beta-lactamase N Inhibitor cell O 22Dr.T.V.Rao MD
  23. 23. THE FIGHT BETA-LACTAMASE INHIBITOR PG beta-lactamase Inhibitor N cell O 23Dr.T.V.Rao MD LYSIS
  24. 24. ESBLSEnterobacteriaceaeResistance to oxyimino-cephalosporins and Monobactamsbut not cephamycins and carbapenem • Susceptible to beta-lactamase inhibitors •GenesSHVTEMCTX-MOXAAmpC 24 Oteo, et al., 2010Dr.T.V.Rao MD
  25. 25. Evolution of -Lactamases Plasmid-mediated TEM and SHV -lactamases Extended-spectrumAmpicillin Cephalosporins 1963 1965 1970s 1983 1988 2000 Look and you will find ESBL TEM-1 TEM-1 ESBL in ESBL > 130 ESBLs E.coli Reported in Europe in USA Worldwide S.paratyphi 28 Gm(-) sp 25 D r .
  26. 26. CLASSIFICATION OF Β LACTAMASESRichards and Sykes (1971) • substrateAmbler (1969) • structureBush, Jacoby, Medeiros (1995) • Substrate; correlation with molecular structure • 150 TEM; • 88 SHV; • 88 OXA, • 53 CTX-M; • 22 IMP; • 12 VIM + smaller number of other enzymes (http://www.lahey.o 26Dr.T.V.Rao MD
  27. 27. CLASSIFICATIONAmbler Classification •Molecular class A – D •ABush-Jacoby-Medeiros Classification •Functional group 1 – 4 •2 • 2b • 2be 27 Paterson and Bonomo, 2005Dr.T.V.Rao MD
  28. 28. BETA-LACTAMASE INHIBITORSResemble β-lactam antibiotic structureBind to β-lactamase and protect theantibiotic from destructionMost successful when they bind the β-lactamase irreversiblyThree important in medicine •Clavulanic acid •Sulbactam •TazobactamDr.T.V.Rao MD
  29. 29. RESISTANCE AND GENETICS AmpC TEM CTX-M K1 Hi-level ESBLCeftazidime R R v SCefotaxime R v R SCefoxitin R S S SAztreonam R v v RSynergy + clav No +++ +++ No 29 Dr.T.V.Rao MD Know the species
  30. 30. Why Test for β-lactamases ? Improve clinical outcome  Inappropriate treatment leads to poor outcome  Each 1 hour delay increases mortality by 7.6% in septic shock1 Encourage antimicrobial stewardship  Spare carbapenems..  Reduce C. difficile / antibiotic associated diarhoea Enhanced surveillance  Identify emerging resistance problems  Develop structures to prevent dissemination Infection Control  ‘Search and Destroy’ analogous to MRSA ? Laboratory Detection is not always easy… OR Rapid1Kumar, Crit Care Med, 2006
  31. 31. TYPES OF ESBLSTEMSHVCTX-M MutationsOXA ESBL Phenotype Plasmid-mediatedDr.T.V.Rao MD
  32. 32. CHOICE OF INDICATOR CEPHALOSPORINTEM & SHV – obvious resistance toceftazidime, variable to cefotaximeCTX-M – obvious resistance tocefotaxime, variable to ceftazidimeAll ESBLs – obvious resistance tocefpodoximeCefuroxime, cephalexin andcephradine are unreliable indicators 32 Livermore D and Woodford N HPA Guidance 2004
  33. 33. CURRENT MODERN METHODS CLSI – Clinical Laboratory and Standards Institute ARMRL - Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency Centre for Infections, London EUCAST- European Society of Clinical Microbiology & Infectious Diseases Commercial methods – Etest, BD Phoenix, Vitek Neo tabs & others 33Slide
  34. 34. DETECTION OF ESBLSSeek ceph/clav synergy in ceph R isolates •Double disc •Combination disc •Etest 34Dr.T.V.Rao MD
  35. 35. CHALLENGES FOR THE DIAGNOSTIC LABDetection…. Hemophilus, Neisseria etc.Predicting -lactamase types. Have GNB got ?:  ESBL, AmpC Metallo types, VIM, IMP etc…Spotting unusual patterns; knowing what to refer ???
  36. 36. DETECTION STRATEGY: STEP 1Screen Enterobacteriaceae with :• Cefpodoxime- best general ESBL substrate• Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively 36Dr.T.V.Rao MD
  37. 37. COMBINATION DISK METHOD CARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 - 4232 Difference > 5 mm 37Dr.T.V.Rao MD
  38. 38. 38Dr.T.V.Rao MD
  39. 39. 39Dr.T.V.Rao MD
  40. 40. KLEBSIELLA PNEUMONIA PRODUCING A HIGHER ACTIVITY ESBLThe higher level ofESBL production isindicated by theinhibition of theβ-lactamase byclavulanic acid andthe resulting ellipticalinhibitory zonebetween cefotaxime(CTX 5) andAugmentin (AMC 60). 40 Dr.T.V.Rao MD
  41. 41. Double disc antagonism for inducible AmpC Cefoxitin Ceftazidime 41 Dr.T.V.Rao MD
  42. 42. AMPC INDUCIBILITY- WHEN TO LOOK Rarely!!!!! Risk is mutation, not inducibility per se Best to identify & predict risk from species Biggest risk Enterobacter & C freundii Avoid cephalosporins against themIdentify means identify TO SPECIES LEVEL allEnterobacteriaceae (‘coliforms’) ex serious infections
  43. 43. ESBLS DETECTION METHODS: INHIBITION BY CLAVULANIC ACID Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and aztreonam discs (30 mcg each)43
  44. 44. ESBL DETECTION – Screen cefpodoxime ; cefotaxime & ceftazidime – Synergy test with ceph/clavCombination discs aremost cost effectivesynergy tests; Etestsa good alternative.. orautomate 44Dr.T.V.Rao MD
  45. 45. ESBL Confirmatory Test Positive for ESBL Cefotax/CA Ceftaz/CA Ceftaz Cefotax 45Dr.T.V.Rao MD
  46. 46. ESBL CONFIRMATORY TEST NEGATIVE FOR ESBL Ceftaz/CA Cefotaxime/CA Ceftaz CefotaxDr.T.V.Rao MD
  47. 47. ESBL CONFIRMATORY TEST Etest Ceftaz/CA Ceftaz 47Dr.T.V.Rao MD
  48. 48. ESBLS: TIMES A’ CHANGING WITH CTX-MOld advice- test ceftazidime; ESBL test if RNew advice- test ceftazidime & cefotaxime;ESBL test if R to either• Alternative- test cefpodoxime; ESBL test if R• Still true- Only testing cefuroxime is inadequate 48Dr.T.V.Rao MD
  49. 49. COMPARING DISK DIFFUSION WITH MINIMUM INHIBITORY CONCENTRATIONS Disk diffusion MICscefpodoxime < 22 mm cefpodoxime > 2 µg/mlceftazidime < 22 mm ceftazidime > 2 µg/mlaztreonam < 27 mm aztreonam > 2 µg/mlcefotaxime < 27 mm cefotaxime > 2 µg/mlceftriaxone < 25 mm ceftriaxone > 2 µg/ml 49Dr.T.V.Rao MD
  50. 50. ESBL CONFIRMATORY TESTSDouble-disk synergy (DDS) test• CAZ and CAZ/CA disks• CTX and CTXCA disks• Confirmatory testing requires using both CAZ and CTX alone and with CA• 5 mm enhancement of the inhibition zone of antibiotic/CA combination vs antibiotic tested alone = ESBL 50 Dr.T.V.Rao MD
  51. 51. SYNERGY TESTS WITH 4-GEN CEPHALOSPORINSCefepime/clav (Mast & AB Biodisk)Cefpirome clav (Oxoid) • Devt. driven by spread of clonal E. aerogenes with TEM-24 in Belgium & France • Sensitivity for weak ESBLs remains to be proven • Cefpirome & cefepime products need 51Dr.T.V.Rao MDcomparison
  52. 52. PITFALLS IN ESBL DETECTION•Methods optimised for E. coli &Klebsiella•More difficult with Enterobacter – clavulanate induces AmpC; hides ESBL•Best advice is to do synergy test (NOTSCREEN) with 4th gen ceph 52 Dr.T.V.Rao MD
  53. 53. RISK FACTORS FOR ESBL INFECTIONLength of hospital staySeverity of illnessTime in the ICUIntubation and mechanical ventilationUrinary or arterial catheterizationPrevious exposure to antibiotics53Dr.T.V.Rao MD
  54. 54. BACTERIA NOT TO TEST FOR ESBL’S Acinetobacter – Often S to clavulanate alone S. maltophilia – +vet result by inhibition of L-2 chromosomal - lactamase, ubiquitous in the species 54Dr.T.V.Rao MD
  55. 55. ESBL REPORTING RULEThe rule (CLSI =NCCLS) M100-S15)… • “Strains of Klebsiella spp. E. coli, and Proteus mirabilis that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents.”The message… • Report “confirmed” ESBL-producing strains as R to all penicillins, cephalosporins, and aztreonam 55Dr.T.V.Rao MD
  56. 56. WILL CLSI CONFIRMATORY TESTDETECT ALL ESBL-PRODUCING GNB?No - some isolates have ESBLs plus otherresistance mechanisms that mask ESBLdetection in the confirmatory test, e.g., • > 1 ESBL • ESBL + AmpC • ESBL + porin mutationESBLs occur in species other than E. coli,Klebsiella spp., and Proteus mirabilis whichCLSI does not currently address 56Dr.T.V.Rao MD
  57. 57. ESBL DETECTION: AUTOMATED SYSTEMS (AS)144 putative of ESBL producersESBL detection: •AS: Microscan, Vitek2, Phoenix •Phenotypic tests: Etest, DDS •Molecular tests: PCR, IsoElectric Focusing (IEF)Molecular identification: the referencemethod 57 Dr.T.V.Rao MD
  58. 58. THE RESISTANCE BECOMING COMPLEXBeta-lactamases are getting more complexFull I/D needs complex molecular methodsMuch can be inferred from simple tests. Needs I/D Testing wide panels of antibiotics; synergy tests Knowledge of what’s unusual 58 Dr.T.V.Rao MD
  59. 59. ANTIBIOTIC POLICY CHANGES PRACTISEDNitrofurantion substituted for quinolonesin UTIsImipenem substituted for quinolones inserious sepsisErtapenem introduced for ESBL sepsisGentamicin substituted forcephalosporins in surgical prophylaxisReturn to amoxycillin in respiratory tractinfections 59 Dr.T.V.Rao MD
  60. 60. MICROBIOLOGY LABORATORIES AND ESBL’SUnfortunately, many clinical laboratorieslack of understanding regarding ESBLsand Ampc ß-lactamase and theirdetection .This has been documented ina study in Connecticut USA, where it wasfound that 21% of laboratories failed todetect extended –spectrumcephalosporins and Aztreonam in ESBLsand Ampc. The true prevalence of ESBLs is notknown and is probably underestimatedbecause of difficulties encounter in theirdetection. However, it is clear that ESBLsDr.T.V.Rao MD
  61. 61. HAND WASHING STILL CAN REDUCE THE ESBL SPREAD 61Dr.T.V.Rao MD
  62. 62. The programme created by Dr.T.V.Rao MD for basic understanding by Medical Microbiologists in the Developing World Email doctortvrao@gmail.com 62Dr.T.V.Rao MD

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