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Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
Extended spectrum Beta Lactamases
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Extended spectrum Beta Lactamases

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Extended spectrum Beta Lactamases

Extended spectrum Beta Lactamases

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  • 1. ESBL DETECTION METHODS DR.T.V.RAO MDDR.T.V.RAO MD 1
  • 2. WHAT ARE EXTENDED-SPECTRUM Β-LACTAMASES?• ESBLs are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and Cefotetan) or carbapenems (e.g., meropenem or imipenem).DR.T.V.RAO MD 2
  • 3. SURVIVAL OF THE FITTEST • Resistant bacteria survive, susceptible ones dieMutant emerges Sensitive cells Mutant’s progeny slowly killed by antibiotic overrun 3
  • 4. WHY SHOULD CLINICAL LABORATORY PERSONNEL BE CONCERNED ABOUT DETECTING THESE ENZYMES?• The presence of an ESBL-producing organism in a clinical infection can result in treatment failure if one of the above classes of drugs is used. ESBLs can be difficult to detect because they have different levels of activity against various cephalosporins. Thus, the choice of which antimicrobial agents to test is critical. For example, one enzyme may actively hydrolyze ceftazidime, resulting in ceftazidime minimum inhibitory concentrations (MICs) of 256 g/ml, but have poor activity on cefotaxime, producing MICs of only 4 g/ml. If an ESBL is detected, all penicillins, cephalosporins, and aztreonam should be reported as resistant, even if in vitro test results indicate susceptibilityDR.T.V.RAO MD 4
  • 5. DEFINITION OF ESBL BL: • 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 includedSLIDE 5
  • 6. Β-LACTAM ANTIBIOTICS • Penicillins • Ampicillin • Piperacillin • Beta-lactam/beta-lactamase inhibitors • Ampicillin/sulbactam • Amoxicillin/clavulanate • Ticarcillin/clavulanate • Piperacillin/TazobactamDR.T.V.RAO MD 6
  • 7. Β-LACTAM ANTIBIOTICS• First Generation cephalosporins • Cefazolin • Cephalothin• Second Generation oral antibiotics • Cefuroxime (many others)• Second Generation cephamycins • Cefoxitin • Cefotetan DR.T.V.RAO MD 7
  • 8. RISK FACTORS FOR ESBL INFECTION• Length of hospital stay• Severity of illness• Time in the ICU• Intubation and mechanical ventilation• Urinary or arterial catheterization• Previous exposure to antibiotics 8 Bradford PA. Clin Microbiol Rev. 2001;14:933-951.
  • 9. Β-LACTAM RESISTANCE IN GRAM NEGATIVE BACTERIA• Ampicillin resistance in Enterobacteriaceae • Acquisition of TEM-1 β-lactamase in E. coli, SHV-1 in K. pneumonia• Cephalosporin resistance developed by mutation of TEM and SHV • Point mutations in TEM and SHV change structure of the enzyme • Enables hydrolysis of cefuroxime, cephalexin, cefadroxil, cephalothin etc…..• Extended spectrum β-lactamases • More TEM and SHV variants and emergence of CTX-M, VEB, PER • Resistance to 3rd and 4th generation cephalosporins (ceftazidime, cefotaxime) DR.T.V.RAO MD 9
  • 10. Β-LACTAM RESISTANCE IN GRAM NEGATIVE BACTERIA • AmpC β-lactamases • Natural β-lactamases able to hydrolyze cephalosporins at low level • Mutations in regulatory genes leave to ‘derepression’ and overexpression in Enterobacter, Serratia, Morganella spp • Carbapenemases – resistance to cephalosporins and carbapenems • Acquired KPC in K. pneumoniae, Enterobacter, E.coli • Zn dependent mettallo-enzymes (IMP, VIM) in P. aeruginosa, A. baumannii DR.T.V.RAO MD 10
  • 11. THERE ARE MORE THAN 200 BETA-LACTAMASE TYPES IN GRAM NEGATIVE BACILLI• Class A: TEM-1,2; SHV-1; ESBLs, KPC• Class B: MBLs• Class C: AmpC• Class D: OXA DR.T.V.RAO MD 11
  • 12. Evolution of β-Lactamases Plasmid-mediated TEM and SHV β-lactamases Extended-spectrum Ampicillin 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
  • 13. CLASSIFICATION OF Β LACTAMASES• Richards and Sykes (1971) • substrate• Ambler (1969) • structure• Bush, 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
  • 14. Evolution of β-LactamasesPlasmid-mediated TEM and SHV β-lactamasesAmpicilli Extended-spectrumn Cephalosporins 1965 1970s 1983 1988 2000 TEM-1 TEM-1 ESBL in ESBL > 130 ESBLs 1963 E.coli Reported in Europe in USA Worldwide S.paratyphi 28 Gm(-) sp DR.T.V.RAO MD 14 14
  • 15. 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 15
  • 16. MODIFIED BUSH–JACOBY–MEDEIROS CCLASSIFICATION OF Β–LACTAMASESFunctional Substrate profile Molecular Inhibitor ExampleGroup Class1 Cephalosporinase C Oxa AmpC, MIR-12a Penicillinase A Clav. S.aureus2b Broad spectrum A Clav. TEM-1/2, SHV-12be Extended spectrum A Clav. TEM 3-29, TEM46-104 SHV2- 28, CTX-M types2br Inhibition resistant A - TEM 30-41 (IRT1-12)2c Carbenicillinase A Clav. PSE-12d Oxacillinase D (Clav.) OXA-1 (OXA-2 &-10 derived ESBL)2e Cephalosporinase A Clav. FPM-1 P. vulgaris, CepA B. fragilis.2f Carbapenemase A Clav. IMI-1, NmcA, Sme 1-33 Metallo-enzyme B - S.maltophilia DR.T.V.RAO MD 164 Penicillinase - - B.cepacia
  • 17. AMPC Β-LACTAMASES • Chromosomally encoded-cell wall turnover • Enterobacter sp., Citrobacter sp., Serratia sp., Morganella sp. Even E. coli. • Third-generation cephalosporins are not good inducers of AmpC β-lactamase • Third-generation cephalosporin resistant strains are derepressed—meaning that the AmpC β- lactamase is not inducible anymore. • AmpC mutants are cephamycin resistantDR.T.V.RAO MD 17
  • 18. AMPC β-LACTAMASES• Molecular class C, functional group 1• Not inhibited by CA• Confers resistance to penicillins, cephalosporins, monobactam, and cephamycin• Chromosomally- or plasmid-mediated• Many genera in Enterobacteriaceae encode chromosomal inducible AmpC• Serratia marcescens• Enterobacter cloacae• Citrobacter freundii• Morganella morganii• Hafnia alvei• Yersenia enterocolitica• Pseudomonas aeruginosa
  • 19. AMPC β-LACTAMASES• Expression of the chromosomal ampC is generally low• Inducible in response to certain β-lactams• Factors involved in ampC induction: • β-lactam interaction with PBPs • Byproducts of cell wall synthesis • Gene products • AmpR • AmpD • AmpG
  • 20. CTX-M• Fast growing – important group• Preferentially hydrolyse, and confer resistance to cefotaxime• Escape of chromosomal ß-lactamase genes from Kluyvera spp (a bug of no clinical importance!)• Having migrated to mobile DNA, CTX-M ß-lactamases genes may evolve further – undergoing mutations that increase activity against ceftazidime• The first CTX-M ESBL in the UK was found as recently as 2000, in a solitary isolate of K. oxytoca• First outbreak, caused by K. pneumoniae producing the new enzyme CTX-M-26, was recorded in Birmingham in 2001 Livermore D and Hawkey P. J Antimicrob Chemother 2005; 56: 451-454 20 HPA Report September 2005 www.hpa.org.uk/publications
  • 21. CTX-M• Has supplanted TEM and SHV types as the predominant ESBLs in the UK• CTX-M-15 enzyme most common in UK• 28/105 cases resulted in death in one UK PCT• Most CTX-M-15 producing E. Coli isolates tested by HPA were multi-resistant to aminoglycosides, fluoroquinolones and trimethoprim as well as all ß-lactams, except carbapenems and temocillin 21 HPA Report September 2005 www.hpa.org.uk/publications
  • 22. INCREASING NUMBERS OF ESBLS 80 70# of ESBLs per year 60 50 40 30 20 10 0 2000 2001 2002 2003 2004 2005 2006 2007 Lewis, et al. AAC 51:4015, 2007 22
  • 23. BETA-LACTAMASE INHIBITORS• Resemble β-lactam antibiotic structure• Bind to β-lactamase and protect the antibiotic from destruction• Most successful when they bind the β-lactamase irreversibly• Three important in medicine • Clavulanic acid • Sulbactam • Tazobactam DR.T.V.RAO MD 23
  • 24. TYPES OF ESBLS• TEM• SHV• CTX-M Mutations• OXA ESBL Phenotype Plasmid-mediated DR.T.V.RAO MD 24
  • 25. ESBLS ARE BETA-LACTAMASES WHICH: • Hydrolyse third generation cephalosporins (and aztreonam, penicillins and many other cephalosporins) • Do not appreciably hydrolyse cephamycins (cefoxitin or Cefotetan) or carbapenems • Are inhibited by beta-lactamase inhibitors such as clavulanic acidDR.T.V.RAO MD 25
  • 26. HISTORICAL PERSPECTIVES LABORATORY DETECTION (V-1) 1988 Jarlier effect – CTX with Augmentin (Jarlier V et al Rev Infect Dis 1988) 1990 NCCLS– ceftazidime zone <15mm Kirby Bauer Method for screening 1994 Synergy testing with ceftazidime (Sader HS et al Diagn Microbiol Infect Dis 1994)SLIDE 26
  • 27. LABORATORY DETECTION OF ESBL• Phenotypic Methods • Screening methods • Confirmatory Methods• Genotypic Methods
  • 28. 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
  • 29. LABORATORY DETECTION 1996 Etest with ceftazidime and clavulanate was recommended (Cormican MG et al JCM) 1996 >50% ESBL E. coli and 29% of ESBL K. pneumoniae were resistant to cefoxitin and 10% of non-ESBL E.coli and K. pneumoniae also resistant to cefoxitin Jacoby GA & Han P JCM ) 2001 Cefpodoxime recommended for screening Clin Microbiol Rev 2001SLIDE 29
  • 30. WHY DETECT ESBL PRODUCERS?• ESBL producers may:• Appear Sensitive to some cephalosporins s in vitro• Show major inoculum effects• Fail in therapy, despite appearing susceptible DR.T.V.RAO MD 30
  • 31. ESBL PRODUCERS FREQUENTLY APPEAR SUSCEPTIBLE TO CEPHALOSPORINS80 • Enterobacteriaceae are70 traditionally reported as60 susceptible to ceftazidime,50 Cefotax. cefotaxime, ceftriaxone,40 Ceftriax. aztreonam, and cefepime30 Ceftaz. when MIC <= 8 µg/mL2010 0 <=8 >=32 (S) (R)
  • 32. CHOICE OF INDICATOR CEPHALOSPORIN• TEM & SHV – obvious resistance to ceftazidime, variable to cefotaxime• CTX-M – obvious resistance to cefotaxime, variable to ceftazidime• All ESBLs – obvious resistance to cefpodoxime• Cefuroxime, cephalexin and cephradine are unreliable indicators 32 Livermore D and Woodford N HPA Guidance 2004
  • 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 & othersSLIDE 33
  • 34. COMMON ESBL PRODUCERS• Klebsiella pneumoniae• Escherichia coli• Proteus mirabilis• Enterobacter cloacae• Non-typhoidal Salmonella (in some countries)DR.T.V.RAO MD 34
  • 35. DIVERSITY OF ESBL’S• Confer resistance to 1st , 2nd, 3rd cef. • Most are susceptible to β-lactamase inhibitors • Most are susceptible to 4th cef. • All are susceptible to carbapenems• Diversity of ESBL • SHV (widespread) • TEM (>100 types) • OXA • Predominantly in Pseudomonas • less susceptible to β-lactamase inhibitors • CTX-M • Probably independent evolution • Highly resistant to 3rd generation cephalosporines • initially in South America, Far East & Eastern Europe • Probably most frequent worldwide
  • 36. DETECTION STRATEGY: STEP 1• Screen Enterobacteriaceae with :• Cefpodoxime- best general ESBL substrate• Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively DR.T.V.RAO MD 36
  • 37. DETECTION OF ESBLS: STEP 2• Seek ceph/clav synergy in ceph R isolates •Double disc •Combination disc •Etest DR.T.V.RAO MD 37
  • 38. COMBINATION DISK METHODCARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 - 4232 Difference > 5 mm
  • 39. 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
  • 40. DR.T.V.RAO MD 40
  • 41. DR.T.V.RAO MD 41
  • 42. ESBLS DETECTION METHODS: INHIBITION BY CLAVULANIC ACIDCo-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime andaztreonam discs (30 mcg each) 42
  • 43. ESBL DETECTION : COMBINATION DISCS: +VE RESULT, ZONE ENLARGED 50%D iscs (30+10 µ g) % D etected (n =100)C eftazidim e +/- clav 88C efotaxim e +/- clav 66B oth 93 M’Zali et al. 2000, JAC, 45, 881 DR.T.V.RAO MD 43
  • 44. ESBL DETECTION•2 Steps: – Screen cefpodoxime ; cefotaxime & ceftazidime – Synergy test with ceph/clav•Combination discs are most cost effective synergy tests; Etestsa good alternative.. or automate•Guidelines on http//www.hpa.org.uk- type ESBL in search facility DR.T.V.RAO MD 44
  • 45. COMPARING DISK DIFFUSION WITH MINIMUM INHIBITORY CONCENTRATIONS Disk diffusion MICscefpodoxime < 22 mm cefpodoxime > 2 g/mlceftazidime < 22 mm ceftazidime > 2 g/mlaztreonam < 27 mm aztreonam > 2 g/mlcefotaxime < 27 mm cefotaxime > 2 g/ml DR.T.V.RAO MD 45
  • 46. Etest for ESBLsCefotaxime Cefotaxime + clavulanate DR.T.V.RAO MD 46
  • 47. ESBL Confirmatory Test Positive for ESBL Cefotax/CA Ceftaz/CA Ceftaz CefotaxDR.T.V.RAO MD 47 47
  • 48. ESBL CONFIRMATORY TEST NEGATIVE FOR ESBL Ceftaz/CA Cefotaxime/CA Ceftaz CefotaxDR.T.V.RAO MD 48 48
  • 49. ESBL CONFIRMATORY TEST Etest Ceftaz/CA CeftazDR.T.V.RAO MD 49 49
  • 50. 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 (NOT SCREEN) with 4th gen ceph DR.T.V.RAO MD 50
  • 51. SYNERGY TESTS WITH 4-GEN CEPHS •Cefepime/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 comparison DR.T.V.RAO MD 51
  • 52. CEPH R BUT SYNERGY –VE…AmpC- plasmid or S to 4 gen cephschromosomalK1 hyperproducer R cefuroxime, aztreonam, cefpodoximeK. oxytoca S ceftazidime, I to cefotaxime May give false +ve ESBL testImpermeable E. R cefoxitin & cefuroxime; not ¾-gen cephscoli, KlebCarbapenemase R includes imipenem & / or meropenemMetallo or not DR.T.V.RAO MD 52
  • 53. BACTERIA NOT TO TEST FOR ESBLS •Acinetobacter – Often S to clavulanate alone •S. maltophilia – +vet result by inhibition of L-2 chromosomal β- lactamase, ubiquitous in the species DR.T.V.RAO MD 53
  • 54. ESBL REPORTING RULE• The 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 DR.T.V.RAO MD 54 54
  • 55. WILL CLSI CONFIRMATORY TEST DETECT ALL ESBL-PRODUCING GNR?• No - some isolates have ESBLs plus other resistance mechanisms that mask ESBL detection in the confirmatory test, e.g., • > 1 ESBL • ESBL + AmpC • ESBL + porin mutation• ESBLs occur in species other than E. coli, Klebsiella spp., and Proteus mirabilis which CLSI does not currently address 55
  • 56. MOLECULAR DETECTION OF ESBLS• PCR and sequencing • The gold standard • Can detect all variants • Easy to perform • Labor intensive
  • 57. ESBL DETECTION: AUTOMATED SYSTEMS (AS)• 144 putative of ESBL producers• ESBL detection: • AS: Microscan, Vitek2, Phoenix • Phenotypic tests: Etest, DDS • Molecular tests: PCR, IsoElectric Focusing (IEF)• Molecular identification: the reference method • JCM. Apr. 2007, p.1167-1174
  • 58. ESBL DETECTION: AUTOMATED SYSTEMSDetection Sensitivity Specificity PPV NPV Method % % % %MicroScan 83.5 72.9 81.6 75.4 Phoenix 98.8 52.2 75 96.6 Vitek2 85.9 78 84.9 79.3 DDS 92.9 96.6 97.5 90.5 Etest 94.1 84.7 89.9 90.9 JCM. Apr. 2007, p.1167-1174
  • 59. PROBLEMATIC ORGANISMS….• ESBLA organisms with AmpC (Enterobacter, Citrobacter, Serratia) • AmpC is induced by calvulanate • Use cefipime in synergy tests• ESBLCARBA • Mettallocarbapenemases (Pseudomonas, Acinetobacter) • Synergy with EDTA • Hodge test• ESBLM • Difficult ! • Boronic acid for plasmidic AmpC• Numerous commercial disc systems • AmpC and ESBL inhibitors
  • 60. MICROBIOLOGY LABORATORIES AND ESBLS• Unfortunately ,many clinical laboratories lack of understanding regarding ESBLs and Ampc ß-lactamase and their detection .This has been documented in a study in Connecticut USA, where it was found that 21% of laboratories failed to detect extended –spectrum cephalosporins and Aztreonam in ESBLs and Ampc.• The true prevalence of ESBLs is not known and is probably underestimated because of difficulties encounter in their detection. However ,it is clear that ESBLs –producing organisms are distributed worldwide and their prevalence is increasing. 60
  • 61. CARBAPENEMS - TREATMENT OF CHOICE FOR SERIOUS INFECTIONS WITH ESBL PRODUCERS• Carbapenems are not hydrolyzed by ESBLs to any great extent• Success rates with carbapenems for ESBL producers consistently exceed 80%, and in no study has the outcome with carbapenems been surpassed [Paterson CID 2004; Bhavnani DMID 2006; Zanetti AAC 2003]
  • 62. HAND WASHING STILL CAN REDUCE THE ESBL SPREADDR.T.V.RAO MD 62
  • 63. Created by Dr.T.V.Rao MD for ‘ ‘e-learning’ resources on implication of misuse of Antibiotics andconsequences for Medical and Paramedical students in Developing World Email doctortvrao@gmail.comDR.T.V.RAO MD 63

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