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3. Extended-Spectrum-β Lactamase (ESBLs)3
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4. History
Pul Ehrlis Salvarsan Gerhard Domagk Sulfanilamid Alexander Fleming Penicillin Ernest Chain and Howard Florey the drug was tested
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5. Classes of antibacterial agents
Basis of the mechanism of action: Inhibitors of cell wall synthesis Inhibitors of cytoplasmic membrane function Inhibitors of protein synthesis Inhibitors of nucleic acid synthesis antimetabolites
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7. Inhibitors of cell wall synthesis
1-Beta- lactams Penicillins,Cephalosporins,Carbapenems, Monobactams Glycopeptides: Vancomycin,teicoplani (active only against G-P)
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8. b-Lactams: Classification (1)
Penicillins
Narrow-spectrum penicillins
Broad-spectrum penicillins
β-lactamase inhibitor combinations
Oxacillin derivatives
Cephalosporins (ATC/WHO 2005 classification)
1st generation: Gram-positive cocci (GPCs), some Gram-negative bacilli (GNBs)
2nd generation: some GNBs, anaerobes
3rd generation: many GNBs, GPCs
4th generation: many GNBs resistant to 3rd generation, GPCs
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9. b-Lactams: Classification (2)
Carbapenems
Imipenem, meropenem, ertapenem
Monobactams
Aztreonam
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12. The β-lactam family of antibiotics
Penicillins
Cephalosporins
Cephamycins
Carbapenems
Monobactams
Benzyl-penicillin
Cephalothin 1st
Cefoxitin
Imipenem
Aztreonam
Methicillin
Cefamandole 2nd
Cefotetan
Meropenem
Ampicillin
Cefuroxime 2nd
Cefmetazole
Ertapenem
Carbenicillin
Cefotaxime 3rd
Mezlocillin
Ceftazidime 3rd
Ticarcillin
Ceftriaxone 3rd
Cefepime 4th
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13. Definitions Narrow-spectrum -lactam agents
Active against G- or G+ bacteria only; e.g., penicillin
Broad-spectrum -lactam agents
Active against G- and G+ bacteria; e.g., ampicillin, 1st generation cephalosporins 13
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14. Definitions (con’t) Extended-spectrum ß-lactam agents
Enhanced activity against G- and some G+ bacteria; e.g., 3rd and 4th generation cephalosporins, carboxy- and ureidopenicillins
Extended-spectrum ß-lactamases (ESBLs)
Enzymes produced by GNR that destroy certain extended-spectrum ß-lactam agents, including 3rd generation cephalosporins 14
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16. Mechanism of action
Binding to penicillin Binding Protein(P B P) this protein are carboxypeptidase ,aminopeptidase and transpeptidase enzymes Inhibition of one or more of this enzymes accumulation of precursor cell wall unit cell's autolytic system to be activated and results finally cell lyses
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17. Resistance to b-Lactams
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18. Resistance
1-Alteration in target site Production of beta- lactamases Genes encoding are found on the chromosome (generally G-N) and on the plasmid (generally G-P )
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19. 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
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20. β-lactamase inhibitors
Clavulonic acid: derived from Streptomyces clavuligerus
Little antibiotic effect in itself
Given in combination with a β -lactam Ab
Function: by binding the β -lactamase enzyme more efficiently than the actual β -lactam
Thus protect the β -lactam Ab from hydrolysis
Not efficient against cephalosporinases
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22. Evolution of -Lactamases
Plasmid-mediated TEM and SHV -lactamases
Extended-spectrum
Cephalosporins
Ampicillin
1965
1970s
1983
1988
2000
This slide presents the evolution/historical development of the classical plasmid-mediated TEM and SHV -lactamases to extended-spectrum -lactamases. Over a 35 year time period, the transformation has been remarkable.
TEM-1
E.coli
S.paratyphi
TEM-1
Reported in
28 Gm(-) sp
ESBL in
Europe
ESBL
in USA
> 130 ESBLs
Worldwide
1963 22
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23. History of GNR resistance
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
1950
1960
1970
1980
1990
2000
Carbapenemases
1964
Cefalotin use
1941
Penicillin use
Early 1980s
3rd generation ceph.
1983 first ESBL detected in Germany
1959
β -lactamase resistant penicillins: Methicillin
1985
Carbapenem (Imipenem)
1960s
Broad spectrum/ extended spectrum penicillins
1976
β –lactamases inhibitors
2005
Tigecycline
1928
Fleming
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24. Ambler Classification of β-Lactamases
Active site
Serine-enzymes
Zinc-enzymes
Nucleotide sequence A C D B
Four evolutionarily distinct molecular classes
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25. Modified Bush–Jacoby–Medeiros Classification of b–Lactamases
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26. Extended-Spectrum -Lactamases (ESBL)
Confers resistance to penicillins and
1st, 2nd and 3rd generation cephalosporins
Inhibited by -Lactamase inhibitors
Most common CTX-M, TEM and SHV
>300 have been identified
Plasmid-mediated, highly mobile
Often associated with resistance genes to aminoglycoside, TMP/SMX and tetracycline
Associated with fluoroquinolone resistance phenotype
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27. Types of ESBLs TEM SHV CTX-M OXA Mutations ESBL Phenotype
Plasmid-mediated
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28. Evolution of TEM Enzymes
MIC (mg/mL) ceftazidime
TEM
glutamine arginine
TEM-12
2.0
glutamine serine
TEM-26
128
lysine serine
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29. Molecular Basis of ESBLs
Enzyme Ceftaz Amino Acid Position
MIC
TEM Glu Arg Glu
TEM-10 > 256 Glu Ser Lys
TEM Glu Ser Glu
TEM Lys Ser Glu
from: Jacoby, IDCNA 11:875, 1997
Slide 18
As mentioned previously, after many years of the study of the molecular variations involved in substrate profile alternations of TEM enzymes, it is now known that clinically resistant variants are the result of amino acid substitutions in one of several well-defined positions or of combinations of two to five of these substitutions. For each position (with some limited examples), the replacement is with a particular amino acid. In the above example, the increase in the MIC to ceftazidime, beginning with the TEM-1 enzyme can be seen and ending with the TEM-26 enzyme with a ceftazidime MIC of 256 µg/ml. While the mutations examined here include positions 104, 162, and 237, there are also mutations at other positions that have been described. The important take-home message is that standard enzymes are only one or two point mutations removed from those that confer high-level resistance.
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30. Other ESBLs CTX-M preferentially hydrolyze cefotaxime.
- found in strains of Salmonella enterrica
- E.coli and other species of Enterobacteriaceae
OXA
Mainly in P.aeroginosa 6
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31. Patrica A Cli Microbiol Rev 14; 2001
Other ESBls (cont,n)
PER-1 :Discovered in P.aeruginosa (turkey)
PER In south America
VEB E.coli (Vietnam)
CME Chrysobacterium meningosepticum( Thailand)
TLA-1 E.coli ( Mexico )
Patrica A Cli Microbiol Rev 14; 2001
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32. ESBls are clinically important
They destroy cephalosporins
Delayed recognition and inappropriate treatment of sever infection
ESBls –mediated resistance is not always obvious to all cephalosporins
Many ESBL produce multi-resistant to non- -lactam antibiotics such as quinolons .aminoglycosides and trimethoprime 13
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33. Why is important to do special tests and follow complex reporting rules for ESBLs?
In vitro tests may be misleading (e.g., may see “S” in vitro but drug may not work!)
Many types of ESBLs with varying susceptibility profiles (TEM  130; SHV  50; CTX-M  30)
Treatment failures (esp. bacteremia) when patient treated with 3rd generation cephalosporins
ESBL-producing isolates can cause nosocomial outbreaks 33
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34. Which microorganisms are ESBls positive
K.pneumoniae and K.oxytoca
E.coli
Enterobacter spp
Salmonella spp
Morganell morganii,Proteus mirabilis
Serratia marcescens
Pseudomonas aeruginosa 16
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35. ESBL Testing Organisms E. coli, Klebsiella spp., Proteus mirabilis
Screen test: Decreased susceptibility to extended-spectrum ß-lactams
Phenotypic confirmatory test: ß-lactam activity restored by ß-lactamase inhibitor (i.e. clavulanic acid) 35
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36. Disk zone (mm) MIC (µg/ml)
ESBL Screening Test
Disk zone (mm) MIC (µg/ml)
*Cefpodoxime  >4**
*Ceftazidime  >1
*Cefotaxime  >1
Ceftriaxone  >1
Aztreonam  >1
* only these drugs appropriate for P. mirabilis
**>1 g/ml for P. mirabilis
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37. ESBL Phenotypic Confirmatory Test
cefotaxime
cefotaxime/clavulanic acid
ceftazidime
ceftazidime/clavulanic acid
Results:
clavulanic acid restores activity of cefotaxime or ceftazidime or both QC
E. coli ATCC 25922; K. pneumoniae ATCC
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38. CLSI Confirmatory Test – Klebsiella, E. coli, P. mirabilis
MIC Test
cefotaxime and ceftazidime +/- 4 μg/ml clavulanate:
> 3 doubling dilution decrease with either drug
Disk Test
cefotaxime and ceftazidime +/- 10 μg clavulanate
> 5 mm zone increase
e.g. ceftazidime 8 μg/ml
ceftazidime + clavulanate 1 μg/ml
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40. ESBL Confirmatory Test
Positive for ESBL
Cefotax/CA
Ceftaz/CA
Ceftaz
Cefotax 40
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41. ESBL Confirmatory Test Negative for ESBL
Ceftaz/CA
Cefotax/CA
Ceftaz
Cefotax 41
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42. E-test for detection of ESBLs
AB Biodisk (Solna,Sweden) has introduced a two sided ESBL E-test strip that contain either a combination of ceftazidime and ceftazidime/clavulani acid or cefotaxime and cefotaxime /clavulanic acid
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43. E-test for detection ESBls
Both strips have a decreasing gradient of ceftazidime or cefotaxime alone on one end and a decreasing gradient of ceftazidime or cefotaxime plus a fixed gradient of clavulanic acid on the other end .A>3log reduction in the MIC of cefotaxime or ceftazidime in the presence of the clavulanicis considered a positive.
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44. ESBL Confirmatory Test
Ceftaz/CA
Ceftaz
Etest
MicroScan – ESBL panel
Vitek – confirmatory test
Vitek 2 - Advanced Expert 44
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45. Proteus mirabilis and ESBLs
Not necessary to test routinely
Uncommon in USA
Significant problems in some locations (e.g., Europe, South America) 45
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46. Proteus mirabilis ESBL testing
Routine screening not recommended
Perform ESBL test when clinically relevant (e.g. bcteremia)
-consult with medical staff to determine to test
-Practical approach ,test sterile body site isolate
Use Standard ESBLs screen test with slight modification
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47. Proteus mirablis ESBL Screen Test
Drug
Disk Screen
(mm)
MIC Screen
(µ g/ml)
Cefpodoxime 17
≥2*
Ceftazidim
≤22 ≥2
Aztreonam NA
Cefotaxime
≤ 27
Ceftriaxone
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48. Proteus mirabilis ESBLs Testing (con’t)
Use standard disk diffusion or MIC ESBL phenotype confirmatory test without modification 39
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49. 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 49
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50. If ESBL screen test is positive, must we do the confirmatory test prior to reporting isolate as an ESBL?
Yes - unless you are confident that the isolate is / is not an ESBL producer based on:
A previous isolate from the patient
An endemic strain in your institution 50
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51. What about testing / reporting for ESBLs on urine isolates?
The suggestion (CLSI M100-S15)…
“The decision to perform ESBL screening tests on all urine isolates should be made on an institutional basis.”
Rationale…
-lactam concentration in bladder is very high and overcomes hydrolysis by ESBL enzymes
The concerns…
Identifying urine from acute cystitis vs. other
Patient with isolate from urine + other sites
Infection control issues 51
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52. 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 52
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53. ESBLs Test QC frequency Disk Diffusion and MIC Methods
Screen Test
Phenotypic Confirmatory
Test
Daily/weekly QC
K.Pneumoniae ATCC700603 OR
E.Coli ATCC 25922
Daily/Weekly QC
K.pneumoniae ATCC
AND
ESBLs gene on plasmid; must store QC strain at-60°C or lower
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54. 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.
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55. Prevalence of ESBLs
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.
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56. ESBls in Europe
ESBLs were first described in 1983 from Germany and England.
In Netherlands less than 1% of E.coli and K.pneumoniae were ESBLs In France and Italy more than 40% strains of K.pneumoniae resistant to Ceftazidime.
It is not known why the prevalence varies so widely in closely related regions.
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57. North America
The first ESBLs –producing organisms reported in The prevalence of ESBLs production among Enterobacteriaceae in the USA ranges from 0 to 25 % with the national average being 3%
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58. Prevalence of ESBLs USA (cont, )
CDC Report from ICUs(1999)
-E.coli 48%
-K.pneumoniae %
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59. Latin America ESBLs appear common in many Latin Americans countries.
The SENETRY study surveillance program among 10,000 bacterial from ten centers showed:
-45% K.pneumoniae ESBLs positive
- 8.5% E.coli ESBLs positive
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60. Asia and Australia
The prevalence of ESBL –producing strains of Enterobacteriaceae varies fro country to country and from species to species'.
- E.coli 5% in Korea and 23.3% in Indonesia .
- Klebsiella spp 48.8% in Korea and 20-40%
Southeast Asia , China and Japan .Outbreaks of infections due to ESBls –producing organisms have been described widely in Australia
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61. Africa and Middle East
Although national surveillance are not lacking ,outbreaks of infections due to ESBL-producing organism have bee noted in some African nations,
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62. Infection Control caused by ESBLs
Isolation measure
- private room, Private bathroom
- Contact isolation (gloves .gown.
- Hand hygiene compliance
- Dedicated equipment (Thermometer, Stethoscope)
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63. Infection control(cont,)
Identification of patients
-patients charts should be” flagged so that contact isolation for ESBL-colonization can be considered on subsequent admissions thorough decontamination for environmental –of –care with routine low-level disinfectant (Quaternary ammonium ,phenol)
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64. Infection Control(cont,)
Antibiotic Use
Antibiotic use protocol should be institute eliminate indiscriminate use of antibacterials and decrease selective pressure for ESBLs producing strains of bacteria
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65. Increasing Numbers of ESBLs
Lewis, et al. AAC 51:4015, 2007
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66. Current ESBL Detection Methods Fail
Routine tests are not designed for ESBL detection
Low level ESBL expression will not be detected by current tests using low inoculum
MIC values and zone sizes of ESBL producers overlap those of susceptible non-ESBL producers
ESBL double disk test may be inaccurate if positioning is suboptimal
ESBL breakpoint methods are limited since MICs for different strains can range over 7 dilutions
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67. Rapidly Increasing Antibiotic Resistance Constitutes One of the Most Important Clinical, Epidemiological and Microbiological Problems of Today
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69. Thank you!
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