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ESBl Dynamics and Detection

ESBl Dynamics and Detection

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ESBl Dynamics and Detection

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  1. ESBlextended spectrum beta lactamasesdynamics and detection Dr.T.V.Rao Dr.T.V.Rao MD

  2. Resistant bacteria survive, susceptible ones die Survival of the fittest Mutant emerges slowly Sensitive cells killed by antibiotic Mutant’s progeny overrun Dr.T.V.Rao MD

  3. PENICILLIN BETA LACTAM RING BETA LACTAMASES enzymes that inactivate the beta-lactam ring CEPHALOSPORIN BETA LACTAM RING Dr.T.V.Rao MD

  4. Action of a b-lactamase S H2O N Inactive penicilloate O COOH S Active penicillin HN O OH COOH Dr.T.V.Rao MD

  5. Basis of Betalactamse Activity • Time-delayed Growth. Beta-lactamase (red) is produced by the central colony, promoting growth of nearby, non-resistant colonies as it deactivates ampicillin (blue). Diffusion of beta lactamase through agar leads to time-delayed growth of non-resistant colonies Dr.T.V.Rao MD

  6. b-lactam antibiotics • Penicillin's • Ampicillin • Piperacillin • Beta-lactam/beta-lactamase inhibitors • Ampicillin/sulbactam • Amoxicillin/clavulanate • Ticarcillin/clavulanate • Piperacillin/Tazobactam Dr.T.V.Rao MD

  7. Penicillins • 1st gen – strep infection (G+) Ex. Penicillin, Cloxacillin • Extended-spectrum – have broader spectrum against G- including E.Coli Ex. Amoxicillin With inhibitor would protect against some beta-lactamase producers Ex. Amoxicillin/Clav • Broad spectrum – many Enterobacteriaceae Ex Piperacillin/Tazobactam Dr.T.V.Rao MD

  8. Some beta-lactamases only inactivate a small number of antibiotics e.g. penicillin • Others have extendedspectrum to all the penicillins and cephalosporins e.g. cefuroxime, ceftriaxone (ESBLs) • In addition may also carry resistance to other antibiotics e.g. ciprofloxacin. Dr.T.V.Rao MD

  9. : 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 Definition of ESBL Dr.T.V.Rao MD

  10. ESBL Evolution • Mid 1980s • Variants of TEM and SHV • Breakdown 3rd generation cephalosporins • Mainly in hospital Klebsiella • Spread world wide Dr.T.V.Rao MD

  11. 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

  12. Ambler Classification of β-Lactamases β-lactamases Active site Serine-enzymes Zinc-enzymes Nucleotide sequence C D B A Four evolutionarily distinct molecular classes Dr.T.V.Rao MD

  13. Abx Penicillin Cephalosporin Monobactam Carbapenem Bacteriocidal What is a beta-lactam? Google Images Dr.T.V.Rao MD

  14. Cephalosporins 4th 3rd 2nd 1st Willey, et al., 2008 Dr.T.V.Rao MD

  15. Cephalosporins-uses • 1st gen: strep, staph, G- including E.coli Ex. Cefazolin • 2nd gen: greater spectrum against G- Ex. Cefoxitin • 3rd gen: even greater activity, combat narrow-spectrum beta-lactamase producers  ESBLs emerged Ex. Ceftazidime • 4th gen: effective against G- bacilli expressing Xm AmpC resistant to 3rd gen Ex. Cefepime Dr.T.V.Rao MD

  16. Others • Monobactams • Monobactams very active against G- including E.coli Ex. Aztreonam • Carbapenems • Carbapenems have an extremely broad spectrum. Cross-reactivity with penicillins or cephalosporins Ex. Imipenam Dr.T.V.Rao MD

  17. The Fight goes on .. • Beta-lactam • Beta-lactamase • Beta-lactamase inhibitor • ESBL Google Images Dr.T.V.Rao MD

  18. Common ESBL producers Klebsiella pneumoniae Escherichia coli Proteus mirabilis Enterobacter cloacae Non-typhoidal Salmonella (in some countries) Dr.T.V.Rao MD

  19. The FightBeta-Lactam PG cell N O LYSIS Dr.T.V.Rao MD

  20. The FightBeta-lactamase PG beta-lactamase cell N O Dr.T.V.Rao MD

  21. The FightBeta-lactamase PG O NH OH cell Dr.T.V.Rao MD

  22. The FightBeta-lactamase inhibitor PG beta-lactamase cell Inhibitor N O Dr.T.V.Rao MD

  23. The FightBeta-lactamase inhibitor PG beta-lactamase Inhibitor cell N O LYSIS Dr.T.V.Rao MD

  24. ESBLs • Enterobacteriaceae • Resistance to oxyimino-cephalosporins and Monobactams but not cephamycins and carbapenem • Susceptible to beta-lactamase inhibitors • Genes • SHV • TEM • CTX-M • OXA • AmpC Oteo, et al., 2010 Dr.T.V.Rao MD

  25. Evolution of -Lactamases Plasmid-mediated TEM and SHV -lactamases Extended-spectrum Cephalosporins Ampicillin 1983 1965 1970s 1988 2000 1963 Look and you will find ESBL TEM-1 E.coli S.paratyphi TEM-1 Reported in 28 Gm(-) sp ESBL in Europe ESBL in USA > 130 ESBLs Worldwide Dr.T.V.Rao MD

  26. 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 Classification of β lactamases Dr.T.V.Rao MD

  27. Classification • Ambler Classification • Molecular class A – D • A • Bush-Jacoby-Medeiros Classification • Functional group 1 – 4 • 2 • 2b • 2be Paterson and Bonomo, 2005 Dr.T.V.Rao MD

  28. 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 Beta-lactamase inhibitors Dr.T.V.Rao MD

  29. Resistance and genetics Know the species Dr.T.V.Rao MD

  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 Rapid 1Kumar, Crit Care Med, 2006

  31. TEM SHV CTX-M OXA Mutations Plasmid-mediated ESBL Phenotype Types of ESBLs Dr.T.V.Rao MD

  32. 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 Choice of Indicator Cephalosporin Livermore D and Woodford N HPA Guidance 2004

  33. 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, VitekNeo tabs & others Current Modern Methods Slide

  34. Detection of ESBLs • Seek ceph/clav synergy in ceph R isolates • Double disc • Combination disc • Etest Dr.T.V.Rao MD

  35. Challenges for the diagnostic lab • Detection…. Hemophilus, Neisseriaetc. • Predicting b-lactamase types. Have GNB got ?: • ESBL, • AmpC • Metallo types, VIM, IMP etc… • Spotting unusual patterns; knowing what to refer ???

  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

  37. Combination disk methodCarter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232 Difference > 5 mm Dr.T.V.Rao MD

  38. Dr.T.V.Rao MD

  39. Dr.T.V.Rao MD

  40. Klebsiella pneumonia producing a higher activity ESBL • The higher level of ESBL production is indicated by the inhibition of the β‑lactamase by clavulanic acid and the resulting elliptical inhibitory zone between cefotaxime (CTX 5) and Augmentin (AMC 60). Dr.T.V.Rao MD

  41. Double disc antagonism for inducible AmpC Cefoxitin Ceftazidime Dr.T.V.Rao MD

  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 them Identify means identify TO SPECIES LEVEL all Enterobacteriaceae (‘coliforms’) ex serious infections

  43. ESBLs Detection Methods: Inhibition by Clavulanic Acid Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and aztreonam discs (30 mcg each) Dr.T.V.Rao MD

  44. ESBL detection • Screen cefpodoxime ; cefotaxime & ceftazidime • Synergy test with ceph/clav • Combination discs are most cost effective synergy tests; Etests a good alternative.. or automate Dr.T.V.Rao MD

  45. ESBL Confirmatory Test Positive for ESBL Cefotax/CA Ceftaz/CA Ceftaz Cefotax 45 Dr.T.V.Rao MD

  46. ESBL Confirmatory Test Negative for ESBL Ceftaz/CA Cefotaxime/CA Ceftaz Cefotax 46 Dr.T.V.Rao MD

  47. ESBL Confirmatory Test Ceftaz/CA Ceftaz Etest 47 Dr.T.V.Rao MD

  48. ESBLs: times a’ changing with CTX-M • Old advice- test ceftazidime; ESBL test if R • New advice- test ceftazidime & cefotaxime; ESBL test if R to either • Alternative- test cefpodoxime; ESBL test if R • Still true- Only testing cefuroxime is inadequate Dr.T.V.Rao MD

  49. Comparing Disk diffusion with minimum inhibitory concentrations Dr.T.V.Rao MD

  50. ESBL Confirmatory Tests Double-disk synergy (DDS) test • CAZ and CAZ/CA disks • CTX and CTX\CA 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 Dr.T.V.Rao MD

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