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This document provides information on extended spectrum beta-lactamases (ESBLs) in 3 sections. It begins by describing the classification of different types of ESBL enzymes including TEM, SHV, CTX-M, OXA, and less common variants. Next, it outlines laboratory detection methods for ESBLs, including phenotypic and genotypic approaches. It concludes by discussing treatment options against ESBL producers such as carbapenems and piperacillin-tazobactam, as well as infection control measures like isolation, surveillance, and restricting broad spectrum antibiotic use.
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- 1. IN THE NAMEOF GOD IN THE NAME OF GOD 1
- 2. january 2013 Hamadan Universityof Medical Sciences Extended Spectrum Beta-Lactamases (ESBLs) Supervisor: Dr. Yousefi By: Seyed Mostafa Hosseini e.mail: [email protected] 2
- 3. 3 Betalactam Antibiotics Penicillins CephalosporinsMonobactams Carbapenems Advantages High effectiveness Low cost Minimal side effects
- 4. MECHANISMS OF RESISTANCETO Β-LACTAM ANTIBIOTICS 4 1. Inactivation of the β–lactam antibiotic by an enzyme (β–lactamase) 2. Production of newer PBP with decreased affinity to the antibiotic 3. Decreased permeability of the antibiotic through the cell envelope due to altered porin channels 4. Active efflux pumpes that throw the antibiotic out of the cell
- 5. 5 One of themost important resistance mechanism is producing Betalactamase enzymes Extended spectrum beta-lactamases (ESBLs ) This enzymes belong to group 2be, 2d of functional classification and class A , D of molecular classification
- 6. 6 Classification of ESBLs: MainGroups: Other ESBLS: OXA PER VEB TLA BES BEL GES SFO
- 7. 7 TEM was firstdiscovered in E coli in a patient named Temoniera in Greece TEM-1 is the most prevalence in this group TEM-2 is a derivative of TEM-1, which possesses an amino acid substitution Phenotypic variation of ESBLs is caused by alteration of amino acid sequence at the active site TEM-type β-lactamases are most often found in E. coli and K. pneumoniae Until May 2011, 190 types of TEM group have been reported
- 8. 8 SHV-1 was describedin 1972 by Pitton in a Klebsiella pneumoniae isolate pit2 SHV refers to “sulfhydryl variable” of active site All SHV variants described to date are derived from blaSHV-1 with one to seven amino acid substitutions There have been reported for 141 types of SHV at the end of May 2011
- 9. 9 Plasmid-mediated ESBLs CTX-M, hydrolyzecefotaxime over than ceftazidime (cefotaximase, Munich) The CTX-M enzymes originated from Kluyvera spp Of environmental, for example K.ascorbata and K. georgina are the progenitors of the CTX-M2 and CTX-M8. Five group of CTX-M are: CTX-M1, -2, -8, -9 and -25. CTX-M are associated with mobile elements such as ISEcp-1 CTX-M 1, 2, 3, 9, 13, 14, 15, 17, 19, 20, 21 At the end of May 2011, 120 types of enzymes have been reported
- 10. 10 They have highhydrolytic activity against oxacillin and cloxacillin and the poorly inhibited by clavulanic acid Not all OXA enzymes are ESBLs; ESBLs are OXA-11, -14, -16, -17, -18, -19, -28, -31, -32, 35 and -45 Several of the OXA-type ESBLs have been derived from OXA-10 (OXA-11, -14, -16, and 17)
- 11. 11 Inhibitor-resistant β-lactamasesare not ESBLs, but they are often discussed with ESBLs because they are also derivatives of the classical TEM- or SHV-type enzymes The amino acid substitutions in IRTs are different from those that are in ESBL phenotype. This variants are called Complex Mutant of TEM (CMT) that are placed in Bush subgroup 2ber IRTs variants are resistant to inhibition by clavulanic acid and sulbactam but They remain susceptible to inhibition by tazobactam IRT enzymes have also been found among SHV mutant and K.pneumoniae isolates. Seven SHV enzymes (-10, -26, -49, -56, -72, -84, -107) with IRT phenotype are known
- 12. 12 PER-1 type β-lactamasewas reported in 1991 from P. aeruginosa isolate in Turkey PER-type enzymes give high level resistance to ceftazidime, cefotexim and aztreonam but low level resistance to other cephalosporins and inhibited by clavulanic acid but not by tazobactam PER-type enzymes have25% -27 % homology to TEM and SHV
- 13. 13 VEB-1 was firstdescribed in an E coli strain isolated in a French hospital from the pus of a 4-month old Vietnamese boy in 1996 It confers high level resistance to ceftazidim, cefotaxim and aztreonam, and inhibited by clavulanic acid The gene encoding VEB-1 was found to be plasmid mediated; such plasmids also confer resistance to non β- lactam antibiotics
- 14. 14 Which was isolatedfrom Chryseobacterium meningosepticum Which was identified in an E. coli isolate from a patient in Mexico in 1993 Was isolated from a S. marcescens strain from a hospital in Brazil in 1996. It confers a high level of resistance to aztreonam and a higher level of resistance to cefotaxime than to ceftazidime and well-inhibited by clavulanate than by tazobactam
- 15. 15 Was identified ina P. aeruginosa strain isolated in Belgium in 2004. It significantly hydrolyses most expanded-spectrum cephalosporins and aztreonam, but not cephamycin or carbapenem. Its activity is well-inhibited by clavulanate but poorly so by tazobactam It is another uncommon ESBL enzyme that is not closely related to any other plasmid-mediated β-lactamase but does show 36% homology to a carbenicillinase from Proteus mirabilis which is highly related to a class A β-lactamase from Serratia fonticola, high level of resistance to cefotaxime than to ceftazidime and well-inhibited by clavulanate
- 16. 16 First reports ofESBLs in the USA in the late 1980s were reported with TEM-type major enzymes appear to be the TEM and SHV types, with a minimal appearance of CTX-M types During the 1980s and 1990s, TEM and SHV were the predominant ESBL type. Currently, CTX-M and TEM are the main types Rates of ESBLs in South America rank amongst the highest in the world, with CTX-M dominant
- 17. 17 In Asia, highrates of ESBL producing Enterobacteriaceae are seen The first reports from Japan and Taiwan suggest the SHV type played a key role early on but, just like in mainland Europe, the rise of the CTX-M genotype has made it the preeminent enzyme, with national and regional variations CTX-M genotype appears to be the most common type in North Africa. There have also been reports of CTX-M K. pneumoniae in Kenya and SHV and TEM-types in South Africa
- 18. 18 1. Hospital stayof more than seven days 2. Exposure to nosocomial isolates 3. Transfer from another health care facility 4. Central venous catheterization 5. Female sex 6. Hemodialysis 7. Diabetes mellitus 8. Antibiotic use (third and fourth generation cephalosporins) for more than a week 9. Travel to endemic countries
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- 20. 20 PhenotyPic methodS Screening teStS: A. DiskDiffusion method: The screening test must be performed on Mueller Hinton agar on standardized inoculum (0.5 McFarland turbidity) and diameter of the zone of inhibition must be carefully measured after 16-18 hours of incubation at 35-37ºC.
- 21. 21 For E. coli,K. pneumoniae, and K. oxytoca, MIC > 8 µg/ml for cefpodoxime or MIC > 2 µg/ml for ceftazidime, aztreonam, cefotaxime, or ceftriaxone is suggestive of ESBL production. For P. mirabilis, MIC > 2 µg/ml for cefpodoxime, ceftazidime, or cefotaxime is suggestive of ESBL production. B. Microbroth dilution method: C. Chromogenic selective medium: Brilliance ESBL agar (Oxoid, UK) ChromID ESBL agar (bioMe´rieux, France)
- 22. 22 eSbL confirmatory methodS: 1.Combined disk: CAZ/CLA – 22 mmCAZ/CLA – 22 mm CAZ – 11 mmCAZ – 11 mm 22 –11 = > 5mm ESBL
- 23. 23 2. Double DiskSynergy Test (DDST): Double Disk Diffusion Test (DDDT) cefotaximecefotaxime Amoxyclav (amoxicillin/clavula nic acid 30/10 µg) Amoxyclav (amoxicillin/clavula nic acid 30/10 µg)
- 24. 24 3. Three dimensional(3D) tests:
- 25. 25 Commericailly available Methodfor ESBL Detection: 1. E test1. E test
- 26. 26 positive ESBL Etest Unreadable Etest ESBL screen test. The MIC of ceftazidime, which should be read on the end of the strip marked “TZ,” cannot be determined because the inhibition ellipse is distorted by the action of clavulanic acid diffusing from the opposite end of the strip.
- 27. 27 Other Commericailly Methodfor ESBL Detection: 1. Sensititre (TREK Diagnostic system) 2. MicroSan (Siemens Medical Solution Diagnostics) 3. Phoenix (BD Diagnostic system) 4. Vitek (BioMerieux)
- 28. 28 Genotypic methods: 1. ConventionalPCR: Materials: DNA template Forward Primer buffer10x Taq Polymerase Reverse Primer dNTP 10mM Primery Denaturation 94ºC, 5min Final Denaturation 94ºC, 1min 30-35 Annealing 50ºC, 1min Primery Extension 72ºC, 1 min Final Extension 72ºC, 8 min
- 29. 29 2. Mutiplex PCR 3.PCR-Restriction Fragment Length Polymorphism (PCR-RFLP): The amplicons generated following PCR are digested by specific restriction endonucleasesand the fragments that are generated are seprated by gel electrophoresis. The size of fragment indicate point mutations whithin the structural gene. 4. Ligase chain reaction (LCR): Using four different sets of primers. The target DNA sequence after denaturation, the primers hibridize to their complementary strands. The thermostable ligase enzym ligates only the primers that are perfectly complementary to their target sequence. The LCR product is detected by an enzymatic reaction using NADPH- alkalin phosphatase.
- 30. 30 treatment options aGainstesBL producers Carbapenems Piperacillin-tazobactam Fluoroquinolones Cefepime
- 31. 31 infection control Hand washingby hospital personnel Isolation of patients colonised or infected with ESBL producers Clinical and bacteriological surveillance of patients admitted to intensive care units and antibiotic cycling, as well as policies of restriction, especially on the empirical use of broad spectrum antimicrobial agents such as the third and fourth generation cephalosporins and imipenem.
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