4/28/2017 AST

اهداف تعريف بتالاكتاماز آشنايي با ارگانيسم هاي توليد كننده انواع بتالاكتاماز نحوه ارزيابي ارگانيسم هاي توليد كننده بتالاكتاماز 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 Penicillin Binding proteins (PBPs) Inner (cytoplasmic) membrane

Inhibitors of peptidoglycan biosynthesis: fosfomycin D-cycloserine ramoplanin tunicamycin, mureidomycin A, liposidomycin B amphomycin bacitracin penicillins (b-lactams) moenomycin vancomycin (glycopeptides)

Peptidoglycan Synthesis “Penicillin binding protein” Note the D-ala-D-ala vancomycin binding site This crosslinking gives stability and strength to the cell wall

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

The Fight Beta-lactam Beta-lactamase Beta-lactamase inhibitor ESBL MBL Already spoke about the beta-lactam antibiotics and how they act in a bacteriocidal manner to lyse the bacterial cells. Beta-lactamases are a form of antibiotic resistance via enzymatic inhibition. They split the amide bond 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 cell N O LYSIS

The Fight Beta-lactamase PG beta-lactamase cell N O

The Fight Beta-lactamase PG O NH OH cell

The Fight Beta-lactamase inhibitor PG beta-lactamase Inhibitor cell N O

The Fight Beta-lactamase inhibitor PG beta-lactamase Inhibitor cell N O LYSIS

-Lactamase Activity C N H R-CONH S COOH CH3 O -lactam Enzyme-Ser-OH

-Lactamase Activity H H S R-CONH C C CH3 O C N CH3 O H COOH HOH Ser Enzyme

تست هاي سريع تشخيص بتا لاکتاماز تست هاي سريع تشخيص بتا لاکتاماز

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 بتالاکتاماز 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 15 mg Penicillin 5 ” Sodium Chloride 1.5 ” Trisodium Citric Acid 0.3 ” Trisodium Phosphate 18 “ Phenol Red

Nitrocefinروش دیسک

تعریف ESBL 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

2 steps: Screening confirmation ESBL فرایند تشخیص 2 steps: Screening confirmation

ESBL فرایند غربال

به وسیله سفالوسپورین هاESBL فرایند غربال Sensitivity Specificity Cefotaxime & ceftazidime Good Cefpodoxime Moderate Cefuroxime Poor Cephalexin or cephradine Cefpirome or Cefepime Choice of indicator cephalosporin

Combination discs Disc with cephalosporin + clavulanic acid Disc with cephalosporin alone

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

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

سفتازيديم+ كلاولانيك سفتازيديم+ كلاولانيك سفتازيديم 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

Report: S/ imipenem (IMP 10) 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 Ceftaz/CA Ceftaz Etest jhindler nltn MW tele #2 2005

ESBL Reporting Rule The message… 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 handler not MW tale #2 2005

Metallo--Lactamases (MBL) First identified in Japan (P. aeruginosa), 1988 Class B, functional group 3 -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)

فرایند غربال MBL Imipenem, Meropenem zone < 22 mm Ertapenem zone < 21mm

روش های تاييد 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

Disc with Imipenem+EDTA 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)

The same Pseudomonas aeruginosa with 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

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