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4/12/2016 AST 1 اهداف • • • • تعريف بتاالكتاماز آشنايي با ارگانيسم هاي توليد كننده انواع بتاالكتاماز نحوه ارزيابي ارگانيسم هاي توليد كننده بتاالكتاماز تفسير نتايج Overview of Antibiotics as Therapeutic Agents • Selective Inhibition/Toxicity – Due to the differences in structure and metabolic pathways – Harm microorganisms, not the host • Four major sites: – – – – Cell wall Ribosomes DNA Cell membrane Mode of Action Gram-negative cell Gram-positive cell Outer membrane Peptidoglycan Peptidoglycan Penicillin Binding proteins (PBPs) Inner (cytoplasmic) membrane Inhibitors of peptidoglycan biosynthesis: fosfomycin D-cycloserineUDP-MurNAc-L-Ala-D-Glu-L-Lys D-Ala DdlB ATP D-Ala-D-Ala MurF ATP tunicamycin, mureidomycin A, liposidomycin B UDP-MurNAcpentapeptide CYTOPLASM UMP MraY MurE MurD MurC L-Lys ATP D-Glu ATP L-Ala ATP D-Ala D-Ala L-Lys D-Glu L-Ala MurNAc P CELL SURFACE P P amphomycin bacitracin peptidoglycan cross-linking MurB MurA NADPH FADH2 PEP ramoplanin MurG UDPGlcNAc P P P UDPMurNAc D-Ala D-Ala L-Lys D-Glu L-Ala MurNAc GlcNAc P P P P MurNAc GlcNAc MurNAc GlcNAc L-Ala L-Ala D-Glu D-Glu L-Lys Ser-Ala L-Lys Ser-Ala D-Ala D-Ala D-Ala D-Ala penicillins (b-lactams) MurM/N Ala-tRNA Ser-tRNA transglycosylase moenomycin UDPGlcNAc D-Ala D-Ala L-Lys Ser-Ala D-Glu L-Ala MurNAc GlcNAc P P P P MurNAc GlcNAc L-Ala D-Glu L-Lys Ser-Ala D-Ala D-Ala vancomycin (glycopeptides) Peptidoglycan Synthesis “Penicillin binding protein” The β-lactam family of antibiotics Penicillins Cephalosporins Cephamycins Carbapenems Benzylpenicillin Cephalothin 1st Cefoxitin Imipenem Methicillin Cefamandole 2nd Cefotetan Meropenem Ampicillin Cefuroxime 2nd Cefmetazole Ertapenem Carbenicillin Cefotaxime 3rd Mezlocillin Ceftazidime 3rd Ticarcillin Ceftriaxone 3rd Cefepime 4th Monobactams Aztreonam The Fight • Beta-lactam • Beta-lactamase • Beta-lactamase inhibitor • ESBL • MBL Google Images Definition of beta lactamases • Beta-lactamases are enzymes produced by some gram-positive and gram-negative bacteria that hydrolyze beta-lactam antibiotics The Fight Beta-Lactam PG O N cell LYSIS The Fight Beta-lactamase PG beta-lactamase O N cell The Fight Beta-lactamase PG O NH OH cell The Fight Beta-lactamase inhibitor PG beta-lactamase O N Inhibitor cell The Fight Beta-lactamase inhibitor PG beta-lactamase Inhibitor O N cell LYSIS b-Lactamase Activity H H S R-CONH C C C N b-lactam CH3 CH3 O COOH Enzyme-Ser-OH b-Lactamase Activity H H S R-CONH O HOH C C C N O H Ser Enzyme CH3 CH3 COOH تست هاي سريع تشخيص بتا الکتاماز تعاريف • Narrow-spectrum beta-lactam agents – Active against G- or G+ bacteria only e.g. penicillin • Broad-spectrum beta-lactam agents – Active against G- and G+ bacteria e.g. ampicillin, first generation cephalosporins تعاريف • Extended-spectrum beta lactam agents – Enhanced activity against G- and some G+ bacteria e.g. 3rd and 4th generation cephalosporins, carboxy- and ureidopenicillins گونه های که تست بتاالکتاماز برای تشخیص مقاومت در آنها سودمند است Enterococcus species Haemophilus influenzae Moraxella catarrhalis Neisseria gonorrhoeae Staphylococcus species Some anaerobic bacteria Interpretation: A positive test indicates resistance to: •Amoxicillin •Ampicillin •Carbenicillin •Mezlocillin •Penicillin •Piperacillin •Ticarcillin روش اسیدومتری Penicillin 15 mg Sodium Chloride 5” Trisodium Citric Acid 1.5 ” Trisodium Phosphate 0.3 ” Phenol Red 18 “ Nitrocefin روش دیسک ESBL تعریف • Extended-spectrum beta-lactamases (ESBLs): – Hydrolyze 3rd and 4th gen cephalosporins and aztreonam – Do not affect cephamycins (2nd gen ceph) or carbapenems – Remain susceptible to beta-lactamase inhibitors – (Ambler class A, Bush group 2) ESBLs positive Bacteria • • • • • • • • Klebcilla pneumonia and K. oxytoca E. coli Enterobacter sp. Salmonella sp. Morganella morganii Proteus mirabilis Serratia marcescens Pseudomonas aeruginosa ESBL فرایند تشخیص 2 STEPS: SCREENING CONFIRMATION فرایند غربال ESBL به وسیله سفالوسپورین هاESBL فرایند غربال Cefotaxime & ceftazidime Cefpodoxime Sensitivity Specificity Good Good Good Moderate Choice of indicator cephalosporinPoor Cefuroxime Cephalexin or cephradine Cefpirome or Cefepime Poor Moderate Poor Poor Good Combination discs Disc with cephalosporin + clavulanic acid Disc with cephalosporin alone ESBL Confirmatory Test Positive for ESBL Ceftaz/CA Ceftaz Cefotax/CA Cefotax ESBL Confirmatory Test Negative for ESBL Ceftaz/CA Ceftaz Cefotaxime/CA Cefotax سفتازيديم +كالوالنيك سفتازيديم سفتازيديم +كالوالنيك سفتازيديم Double discs synergy test Disc with cephalosporin Disc with clavulanic acid Controls for ESBL tests • +ve E. coli with TEM-3, -10 & CTX-M-15 available as NCTC 13351, 13352, 13353 No one control is perfect… and these have high levels of enzyme whilst some clinical isolate have very low levels • –ve E. coli (e.g. NCTC 10418) Critical for combination discs; should give equal zones irrespective of clavulanate Disc positions recommended for urine isolates Klebsiella pneumoniae producing an ESBL in routine CDS test S/ Augmentin (AMC 60), typical synergy between Augmentin and cefotaxime (CTX 5) / cefepime (FEP 10) Report: S/ imipenem (IMP 10) ESBL Confirmatory Test Etest Ceftaz/CA Ceftaz 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 penicillin's, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents.” • The message… – Report “confirmed” ESBL-producing strains as R to all penicillin's, cephalosporins, and aztreonam Metallo-b-Lactamases (MBL) • • • • • • First identified in Japan (P. aeruginosa), 1988 Class B, functional group 3 b-lactamases Requires Zn2+ for activity Inhibited by EDTA but not by CA Chromosomally or plasmid mediated Broad substrate spectrum including penicillins, cephalosporins, and carbapenemases MBLs • Do not hydrolyze aztreonam • Most common in P. aeruginosa, A. baumannii, and then Enterobacteriaceae • The most common MBL families are: – The largest group: Imipenemases (IMP) – The second largest group: Verona imipenemases (VIM) – German imipenemases (GIM) – Seoul imipenemases (SIM) MBL فرایند غربال • Imipenem, Meropenem zone < 22 mm • Ertapenem zone < 21mm روش های تایید MBL MBL Detection • Different combinations of antibiotics and inhibitors to detect MBL producers with different sensitivity and specificity – Imipenem-EDTA: P. aeruginosa and A. baumannii – Ceftazidime-CA/EDTA: K. pneumoniae – Cefepime-CA/EDTA: E. cloacae and C. freundii Combination discs Disc with Imipenem+EDTA Disc with Imipenem Double discs synergy test Disc with Imipenem Disc with EDTA MBL Detection Disk Approximation Test EDTA 7-mm increase of inhibition zone= MBL Pseudomonas aeruginosa candidate for MBL detection: No zone around imipenem (IPM 10) ceftazidime (CAZ 10), tazocin (TZP 55), cefepime (FEP 10) and Timentin (TIM 85) S/ aztreonam (ATM 30) EDTA 415 EDTA 415 EDTA 415 The same Pseudomonas aeruginosa with EDTA Detection of MBL: Synergy between an EDTA disc placed next to imipenem (IPM 10)/ meropenem (MEM 5)/ ceftazidime (CAZ 10) discs. S/ aztreonam (ATM 30) MBL Detection • Etest: A reduction in the MIC of imipenem of 3 dilution in the presence of EDTA is interpreted as positive Imipenem + EDTA Imipenem Klebsiella pmeumoniae: Synergy between EDTA (blank discs) and IPM 10/ETP 10 only R/ ATM and synergy with AMC 60 => MBL and ESBL Questions?