jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

Extended-Spectrum-β Lactamase (ESBLs) 3 jhindler nltn MW tele #2 2005

History Pul Ehrlis 1910 Salvarsan Gerhard Domagk 1935 Sulfanilamid Alexander Fleming 1929 Penicillin Ernest Chain and Howard Florey 1941 the drug was tested jhindler nltn MW tele #2 2005

Classes of antibacterial agents Basis of the mechanism of action: 1-Inhibitors of cell wall synthesis 2-Inhibitors of cytoplasmic membrane function 3-Inhibitors of protein synthesis 4-Inhibitors of nucleic acid synthesis 5-antimetabolites jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

Inhibitors of cell wall synthesis 1-Beta- lactams Penicillins,Cephalosporins,Carbapenems, Monobactams 2-Glycopeptides: Vancomycin,teicoplani (active only against G-P) jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

b-Lactams: Classification (2) Carbapenems Imipenem, meropenem, ertapenem Monobactams Aztreonam jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Resistance to b-Lactams jhindler nltn MW tele #2 2005

Resistance 1-Alteration in target site 2-Production of beta- lactamases Genes encoding are found on the chromosome (generally G-N) and on the plasmid (generally G-P ) jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Ambler Classification of β-Lactamases Active site Serine-enzymes Zinc-enzymes Nucleotide sequence A C D B Four evolutionarily distinct molecular classes jhindler nltn MW tele #2 2005

Modified Bush–Jacoby–Medeiros Classification of b–Lactamases jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Types of ESBLs TEM SHV CTX-M OXA Mutations ESBL Phenotype Plasmid-mediated jhindler nltn MW tele #2 2005

Evolution of TEM Enzymes MIC (mg/mL) ceftazidime 102 162 TEM-1 0.25 glutamine arginine TEM-12 2.0 glutamine serine TEM-26 128 lysine serine jhindler nltn MW tele #2 2005

Molecular Basis of ESBLs Enzyme Ceftaz Amino Acid Position MIC 104 164 240 TEM-1 0.12 Glu Arg Glu TEM-10 > 256 Glu Ser Lys TEM-12 16 Glu Ser Glu TEM-26 256 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. jhindler nltn MW tele #2 2005 18

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 jhindler nltn MW tele #2 2005

Patrica A Cli Microbiol Rev 14; 2001 Other ESBls (cont,n) PER-1 :Discovered in P.aeruginosa (turkey) PER-2 In south America VEB-1 E.coli (Vietnam) CME-1 Chrysobacterium meningosepticum( Thailand) TLA-1 E.coli ( Mexico ) Patrica A Cli Microbiol Rev 14; 2001 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Disk zone (mm) MIC (µg/ml) ESBL Screening Test Disk zone (mm) MIC (µg/ml) *Cefpodoxime 17 >4** *Ceftazidime 22 >1 *Cefotaxime 27 >1 Ceftriaxone 25 >1 Aztreonam 27 >1 * only these drugs appropriate for P. mirabilis **>1 g/ml for P. mirabilis jhindler nltn MW tele #2 2005

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 700603 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

ESBL Confirmatory Test Positive for ESBL Cefotax/CA Ceftaz/CA Ceftaz Cefotax 40 jhindler nltn MW tele #2 2005

ESBL Confirmatory Test Negative for ESBL Ceftaz/CA Cefotax/CA Ceftaz Cefotax 41 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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. jhindler nltn MW tele #2 2005

ESBL Confirmatory Test Ceftaz/CA Ceftaz Etest MicroScan – ESBL panel Vitek – confirmatory test Vitek 2 - Advanced Expert 44 jhindler nltn MW tele #2 2005

Proteus mirabilis and ESBLs Not necessary to test routinely Uncommon in USA Significant problems in some locations (e.g., Europe, South America) 45 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Proteus mirabilis ESBLs Testing (con’t) Use standard disk diffusion or MIC ESBL phenotype confirmatory test without modification 39 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 700603 AND ESBLs gene on plasmid; must store QC strain at-60°C or lower jhindler nltn MW tele #2 2005

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. jhindler nltn MW tele #2 2005

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. jhindler nltn MW tele #2 2005

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. jhindler nltn MW tele #2 2005

North America The first ESBLs –producing organisms reported in 1988.. The prevalence of ESBLs production among Enterobacteriaceae in the USA ranges from 0 to 25 % with the national average being 3% jhindler nltn MW tele #2 2005

Prevalence of ESBLs USA (cont, ) CDC Report from ICUs(1999) -E.coli 48% -K.pneumoniae 10.4% jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

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, jhindler nltn MW tele #2 2005

Infection Control caused by ESBLs Isolation measure - private room, Private bathroom - Contact isolation (gloves .gown. - Hand hygiene compliance - Dedicated equipment (Thermometer, Stethoscope) jhindler nltn MW tele #2 2005

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) jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Increasing Numbers of ESBLs Lewis, et al. AAC 51:4015, 2007 jhindler nltn MW tele #2 2005

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 jhindler nltn MW tele #2 2005

Rapidly Increasing Antibiotic Resistance Constitutes One of the Most Important Clinical, Epidemiological and Microbiological Problems of Today jhindler nltn MW tele #2 2005

jhindler nltn MW tele #2 2005

Thank you! jhindler nltn MW tele #2 2005