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EXTENDED SPECTRUM B-LACTAMASES (esbl) basis, detection and reporting

EXTENDED SPECTRUM B-LACTAMASES (esbl) basis, detection and reporting. Dr.T.V.Rao MD. V. cholera. C. jejuni. Helicobacter pylori. Acinetobacter spp. Gram Negative Bacilli . Many other ( H. influenza , etc..). Stenotrophomonas maltophilia. Pseudomonas aeruginosa. Enterobacteriaceae.

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EXTENDED SPECTRUM B-LACTAMASES (esbl) basis, detection and reporting

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  1. EXTENDED SPECTRUM B-LACTAMASES(esbl)basis, detection and reporting Dr.T.V.Rao MD Dr.T.V.Rao MD

  2. V. cholera C. jejuni Helicobacter pylori Acinetobacter spp. Gram Negative Bacilli Many other (H. influenza, etc..) Stenotrophomonas maltophilia Pseudomonas aeruginosa Enterobacteriaceae Dr.T.V.Rao MD

  3. 1950 1960 1970 1980 1990 2000 History of resistance in gram-negative bacteria 1965 Broad spectrum β–lactamases (TEM-1 in E. coli) ESBL outbreaks in France 1940 Penicillinase detected in E. coli 1983 Extended spectrum β-lactamases TEM-1 widespread Carbapenemases 1964 Cephalothin use 1941 Penicillin use Early 1980s 3rd generation ceph. 1959 β-lactamase resistant penicillin's: Methicillin 1985 Carbapenems (Imipenem) 1960s Broad spectrum/ extended spectrum penicillin's 1976 β–lactamases inhibitors 2005 Tigecycline 1928 Fleming Dr.T.V.Rao MD

  4. 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

  5. Committees controlling resistance patterns BSAC United Kingdom SRGA Sweden CA-SFM France CRG Netherlands DIN Germany NWGA Norway CLSI (NCCLS) USA Dr.T.V.Rao MD

  6. Standardisation Dr.T.V.Rao MD • 1959 Ericsson & Steers – evaluation of methods • 1961 WHO – standardisation • 1964 Isenberg – comparison of methods in USA • 1964 Truant – standardised tube dilution MICs • 1966 Bauer-Kirby • 1975 NCCLS  CLSI • 1998 BSAC Standardised Method • 2009 EUCAST Standardised Method

  7. Growing incidence of Esbl producers Dr.T.V.Rao MD

  8. Molecular class: A: TEM SHV other B: Metalloenzymes (carbapenemases) C: Prototype: chromosomal ampC D: OXA (oxacillin hydrolyzing enzymes) Enzyme type (by substrate profile): Penicillinase Broad-spectrum Extended Spectrum Carbapenemase Genetic classification: plasmids mediated Chromosomal β-lactamases classification http://www.lahey.org/studies/webt.asp Dr.T.V.Rao MD

  9. β-lactamases Penicillinase: gene blaZ , inducible, on transposon (can move between chromosome and plasmid). Broad spectrum β-lactamases (plasmid encoded) TEM SHV OXA (mainly in pseudomonas) ESBLs TEM related SHV related OXA related CTX-M Other ampCβ-lactamases Resistant to β-lactamase inhibitors chromosomal Carbapenemases Metallo- β-lactamases Serine carbapenemases Types of β-lactamases Dr.T.V.Rao MD

  10. Mutations of TEM-1, TEM-2, SHV-1 Inactivate -lactams with an oxyimino group (third-generation cephalosporins and aztreonam) Plasmid-mediated (often other R genes) 70 TEM and 15 SHV types Extended-Spectrum -lactamases ESBLs Dr.T.V.Rao MD

  11. Broad spectrum b-lactamase (blaTEM) ESBL (TEM related) & & Mutation Plasmid transfer Genetic Mechanism Penicillinase blaZ Transformation Dr.T.V.Rao MD

  12. H H S R-CONH C C CH3 C N CH3 O COOH -Lactamase Activity -lactam Enzyme-Ser-OH Dr.T.V.Rao MD

  13. -Lactamase Activity H H S R-CONH C C CH3 O C N CH3 O H COOH HOH Ser Enzyme Dr.T.V.Rao MD

  14. L      L   L         L    L  L     L    b-lactamase production L  L  Dr.T.V.Rao MD

  15. What are ESBL producing bacteria. • The term ESBLs is used to mean acquired, class A β-lactamases that hydrolyze and confer resistance to oxyimino- ‘2nd- and 3rd-generation’ cephalosporins, eg cefuroxime, cefotaxime, ceftazidime and ceftriaxone Dr.T.V.Rao MD

  16. Confer resistance to 1st , 2nd, 3rd cephalosporins. Most are susceptible to β-lactamase inhibitors Most are susceptible to 4th cephalosporins 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 cephalosporins initially in South America, Far East & Eastern Europe Probably most frequent worldwide Clonal spread has been documented ESBL Dr.T.V.Rao MD

  17. Further complicating matters: • More than one gene of β-lactamase / ESBL / ampC / carbapenemase can be carried on the same plasmid. • Genes of ESBL are carried on plasmids that usually carry additional resistant genes: frequently MDR • Laboratory diagnosis confusing: susceptibility profiles sometimes misleading: “hidden resistance” -> CLSI guidelines are changing. • CTX-M clones appearing in the community (Canada, Greece, Spain, Italy). Dr.T.V.Rao MD

  18. ESBLs include: • Cephalosporin-hydrolysing mutants of TEM and SHV - the common plasmid-mediated penicillinases of Enterobacteriaceae. Well over 100 such variants are known • • CTX-M types. These evolved separately, at least some of them via the escape and mutation of chromosomal β-lactamases of Kluyvera species. Over 30 variants are known Obscure types, e.g. VEB and PER, not yet of concern in the UK; also OXA (Class D) ESBLs from Pseudomonas aeruginosa, in Turkey. Dr.T.V.Rao MD

  19. Esbl are important cause of resistance • ESBLs are not the sole β-lactamases to confer resistance to 2nd and 3rd generation cephalosporins, but are the most important. They occur mostly in Enterobacteriaceae (e.g. E. coli, Klebsiella species and Enterobacter species) and rarely in non-fermenters (e.g. P. aeruginosa). Dr.T.V.Rao MD

  20. They should be distinguishedfrom other important modes of resistance to 2nd and 3rd generation cephalosporins, eg: • Hyper produced chromosomal AmpC β-lactamases, especially in Enterobacter species. • • Plasmid-mediated AmpC β-lactamases, in Klebsiella spp. and E. coli (rare) • • Hyper produced K1 chromosomal β-lactamases in K. oxytoca not pneumoniae) • • Efflux-mediated resistance in P. aeruginosa • • Various ill-defined mechanisms in Acinetobacter species. Dr.T.V.Rao MD

  21. LABORATORY DETECTION: SCREENING andCONFIRMATION Dr.T.V.Rao MD

  22. Primary testing for Esbl detection • The basic strategy to detect ESBL producers is to use an indicator cephalosporin to screen for likely producers, then to seek cephalosporin/clavulanate synergy, which distinguishes ESBL producers from, for example, strains that hyper producer AmpC or K1 enzymes. Dr.T.V.Rao MD

  23. SCREENING • The ideal indicator cephalosporin is one to which all ESBLs confer resistance, even when their production is scanty. Choice is predicated by the following general traits: • TEM and SHV ESBLs – obvious resistance to ceftazidime, variable to cefotaxime • CTX-M ESBLs – obvious resistance to cefotaxime: variable to ceftazidime • All ESBLs – obvious resistance to cefpodoxime Dr.T.V.Rao MD

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  25. Screen Enterobacteriaceae with : Cefpodoxime- best general ESBL substrate Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively Detection Strategy: step 1 Spread of CTX-M into community means screening must be wider than before See http://www.hpa.org.uk Dr.T.V.Rao MD

  26. Seek ceph/clav synergy in ceph R isolates Double disc Combination disc Etest Detection of ESBLs: step 2 See http://www.hpa.org.uk Dr.T.V.Rao MD

  27. 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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  30. Clavulanate Inhibition with Cefotaxime - + + - Ceftazidime - + - + ESBL Confirmation NOT ESBL report results as they appear ESBL report cephalosporins, penicillins, aztreonam as “R” Dr.T.V.Rao MD

  31. CAZ+CA CTX+CA Test for E. coli K. pneumoniae K. oxytoca CMZ CTX CAZ Dr.T.V.Rao MD

  32. ESBL Confirmatory Test Positive for ESBL Cefotax/CA Ceftaz/CA Ceftaz Cefotax Dr.T.V.Rao MD 33

  33. ESBL Confirmatory Test Negative for ESBL Ceftaz/CA Cefotaxime/CA Ceftaz Cefotax Dr.T.V.Rao MD 34

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  38. MIC (g/ml) Cefotaxime >32 Ceftazidime 0.5 Cefotaxime/clavulanate 0.5 Ceftazidime/clavulanate 1.0 ESBL Example Dr.T.V.Rao MD

  39. Ampicillin R Amoxicillin/clavulanate S Cefazolin R Cefotiam R Cefmetazole S Cefotaxime I R Aztreonam R , NCCLS not recommended Cefepime S (?) R Gentamicin R Amikacin S Imipenem S Ciprofloxacin S Flomoxef S ? How to ReportKlebsiella pneumoniae (ESBL) Dr.T.V.Rao MD

  40. It follows that the logical indicator is either cefpodoxime or BOTH of cefotaxime and ceftazidime resistance. • An alternative strategy has been proposed for community urines: testing cephalexin or cephradine as the indicator drug, then doing confirmatory ESBL tests on all isolates that are found resistant (these include e.g. all Enterobacter species. and some hyper producers of classical TEM, as well as the ESBL producers). This is not recommended, as some CTX-M-15 producers, Dr.T.V.Rao MD

  41. Gene on chromosome Produced by virtually all GNBs Activity generally low NOT inhibited by -lactamase inhibitors Differ in E. coli/Klebsiella versus other GNBs AmpC Dr.T.V.Rao MD

  42. Chromosomal Produced in minimal amount NOT inducible AmpC -lactamaseE. coli/Klebsiella spp.-Typical Dr.T.V.Rao MD

  43. AmpC genes mutation Low amount-destroy ampicillin, 1st cephalosporins High amount-destroy expanded spectrum -lactams Transfer of ampC to plasmid Hyper production Resistant to 3rd cephalosporins, cephamycins AmpC -lactamaseE. coli/Klebsiella spp.- Genetic Changes Dr.T.V.Rao MD

  44. Chromosomal Produced in small amount Inducible (hyper production) Reversible AmpC -lactamaseOther GNBs-Typical Dr.T.V.Rao MD

  45. Etest for ESBLs Cefotaxime Cefotaxime + clavulanate Dr.T.V.Rao MD

  46. Etest for ESBLs Cefotaxime Cefotaxime + clavulanate Dr.T.V.Rao MD

  47. Quality control in esbl detection Dr.T.V.Rao MD

  48. CONTROLS FOR ESBL TESTS • Positive controls should be used to ensure the performance of ESBL confirmatory tests. Three ESBL-positive E. coli strains are available from the NCTC: • CTX-M-15 (cefotaximase) NCTC 13353 TEM-3 (broad-spectrum) NCTC 13351 TEM-10 (ceftazidimase) NCTC 13352 Dr.T.V.Rao MD

  49. Clsi recommends • The CLSI recommends K. pneumoniae ATCC 700603 as an ESBL-producing QC control, as does AB Biodisk (Etest). This strain may be sourced from the ATCC. • Either E. coli NCTC 10418 or ATCC 25922 should also be used as a negative control in ESBL confirmation tests. Use of such controls is especially important when the cephalosporin and cephalosporin + clavulanate combination discs are from different batches, which may vary in original content or retained potency. Dr.T.V.Rao MD

  50. Zone determination guides … • Zones of the cephalosporin and cephalosporin + clavulanate discs for ESBL-negative E. coli should be equal or, at worst, within + 2 mm. Any greater difference implies malfunction or deterioration. Dr.T.V.Rao MD

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