Laboratory Identification And Resistance Detection of The Major Pathogenic Gram Negative Organisms

Transcription

1 Laboratory Identification And esistance Detection of The Major Pathogenic Gram Negative Organisms

2 Gram Negative ods 1) E.coli 2) Klebsiella 3) Enterobacter 4) Proteus- Morganella 5) Serratia 6) Pseudomonas 7) Acinetobacter 8) Burkholderia 9) Stenotrophomonas 2

3 Oxidase Positive Non-fermenters Pseudomonas strong + Acinetobacter - Burkholderia slow & weak + Stenotrophomonas - Negative Fermenters Enterobacteriaceae E.Coli Klebsiella Enterobacter Proteus Morganella 3

4 Should use a highly selective gram negative media such as: MacConkey- EMB: E.coli - Klebsiella fermenter Lactose Enterobacter Citrobacter Slow lactose fermenter Others Non lactose fermenter 4

5 Acinetobacter vs. Klebsiella Pseudomonas vs. Burkholderia 5

6 Should use chemical ID schemes to identify Citrate Indole H 2 S production VP M Chromogenic agars Automated ID systems. Visual system: api systems, crystal 6

7 esistant Issues ESBL Carbapenems resistance 7

8 ESBL: Extended Spectrum β-lactamases Enzymes that mediate resistance to: Extended spectrum (3 rd generation) cephalosporins: Ceftazidime, Cefotaxime, Ceftriaxone Monobactams: Azetronam But do not affect Cephamycins: Cefoxitin, Cefotetan Carbapenems: Imipenem, Meropenem, Ertapenem 8

9 3 genes located on plasmid are responsible for ESBL: CTXM, SHV, TEM Often ESBL isolates are multi drug resistant to other classes such as aminoglycosides and quinolones. 9

10 How to screen for ESBL I. Disk diffusion method (KB) II. MIC by automated system ABX Disk diffusion mm MICs µg/ml Cefpodoxime 22 2 Ceftazidime 22 2 Cefotaxime 27 2 Ceftazidime 25 2 Azetronam

11 Susceptibility profile for ESBL Antimicrobial Interpretation Antimicrobial Interpretation Amikacin S / I / Chloramphenicol Amox/clav Ciprofloxacin Ampicillin Ertapenem S Aztreonam Gentamicin Cefazolin Imipenem S Cefpodoxime Meropenem S Cefotaxime Pipercillin/Tazo S / I / Cetotetan Tobramycin Cefoxitin Trimeth/Sulfa Ceftriaxone Tigecycline S Cefepime Ceftazidime 11

12 How to confirm ESBL 1. Synergy test Ceftazidime Cefotaxime Ceftazidime +Clavulunic acid Cefotaxime + Clavulunic acid Not ESBL True ESBL Should see an increase > 5 mm of zone of inhibition to confirm. 12

13 2. ESBL E-test confirmation by 2-gradient strips 13

14 3. Automated system that have a built-in confirmation test 4. Additional strips or panels that test cephalosporins alone and with β-lactamase inhibitors and test for MIC fold differences according to each system`s parameters ( 3 fold increase- 4 fold increase ). 14

15 Mullti Drug esistant Non-fermenter ABX P. Aeroginosa Burkholderia Cepacia Stentrophomonas Acinetobacter Ticarcillin Piperacillin Ampicillin+Sulbactam Ticarcillin+Clav.Ac S / Piperacillin+Clav.Ac S / Imipenem Meropenem S / Ceftazidim S / S / Cefepim S / Amikacin Gentamicin Tobramicin Ciprofloxacin S S / Cotrimoxazol S S Colistin S / S Tigecyclin NA NA S S 15

16 Tigecyclin E-test for Acinetobacter 16

17 17

18 Carbapenems esistance 18

19 Drug Carbapenems oute of Administration FDA Status Imipenem IV Cleared Meropenem IV Cleared Ertapenem IM, IV Cleared Doripenem IV Application Submitted 19

20 Carbapenem esistance: Mechanisms Enterobacteriaceae Cephalosporinase + porin loss Carbapenemase P. aeruginosa Porin loss Up-regulated efflux Carbapenemase Acinetobacter spp. Cephalosporinase + porin loss Carbapenemase 20

21 Carbapenemases Classification Enzyme Most Common Bacteria Class A Class B (metallo-b-lactamse) KPC, SME, IMI, NMC, GES IMP, VIM, GIM, SPM Enterobacteriaceae (rare reports in P. aeruginosa) P. aeruginosa Enterobacteriacea Acinetobacter spp. Class D OXA Acinetobacter spp. 21

22 Carbapenemases in the U.S. Enzyme KPC Metallo-b-lactamase OXA SME Bacteria Enterobacteriaceae P. aeruginosa Acinetobacter spp. Serratia marcesens 22

23 Klebsiella Pneumoniae Carbapenemase KPC is a class A b-lactamase Confers resistance to all b-lactams including extendedspectrum cephalosporins and carbapenems Occurs in Enterobacteriaceae Most commonly in Klebsiella pneumoniae Also reported in: K. oxytoca, Citrobacter freundii, Enterobacter spp., Escherichia coli, Salmonella spp., Serratia spp., Also reported in Pseudomonas aeruginosa (Columbia) 23

24 Susceptibility Profile of KPC-Producing K. pneumoniae Antimicrobial Interpretation Antimicrobial Interpretation Amikacin I Chloramphenicol Amox/clav Ciprofloxacin Ampicillin Ertapenem Aztreonam Gentamicin Cefazolin Imipenem Cefpodoxime Meropenem Cefotaxime Pipercillin/Tazo Cetotetan Tobramycin Cefoxitin Trimeth/Sulfa Ceftazidime Polymyxin B MIC >4mg/ml Ceftriaxone Colistin MIC >4mg/ml Cefepime Tigecycline S 24

25 KPC Enzymes Located on plasmids; conjugative and nonconjugative bla KPC is usually flanked by transposon sequences bla KPC reported on plasmids with: Normal spectrum b-lactamases Extended spectrum b-lactamases Aminoglycoside resistance 25

26 KPC s in Enterobacteriaceae Species Klebsiella spp. Enterobacter spp. Comments K. pneumoniae-cause of outbreaks K. oxytoca-sporadic occurrence Escherichia coli Salmonella spp. Sporadic occurrence Citrobacter freundii Serratia spp. Pseudomonas aeruginosa Columbia & Puerto ico 26

27 Laboratory Detection of KPC- Problems: Producers 1)Some isolates demonstrate low-level carbapenem resistance 2) Some automated systems fail to detect low-level resistance 27

28 No. of Isolates Susceptibility of KPC-Producers to Imipenem Imipenem S* I >32 MIC (mg/ml) *12% of isolates test susceptible to imipenem

29 No. of Isolates Susceptibility of KPC-Producers to Meropenem Meropenem 100 S* I >16 MIC (mg/ml) *9% of isolates test susceptible to meropenem

30 No. of Isolates Susceptibility of KPC-Producers to Ertapenem C-Producers to Ertapenem 60 S I >16 MIC (mg/ml) None of the isolates test susceptible to ertapenem

31 When to Suspect a KPC- Producer Enterobacteriaceae especially Klebsiella pneumoniae that are resistant to extendedspectrum cephalosporins: MIC range for 151 KPC-producing isolates Ceftazidime Ceftriaxone Cefotaxime 32 to >64 mg/ml 64 mg/ml 64 mg/ml Variable susceptibility to cefoxitin and cefepime 31

32 eading Disk Diffusion & Etest 32

33 Phenotypic Tests for Carbapenemase Activity Modified Hodge Test 100% sensitivity in detecting KPC; also positive when other carbapenemases are present 100% specificity 33

34 Modified Hodge Test Lawn of E. coli ATCC :10 dilution of a 0.5 McFarland suspension Test isolates 34 Imipenem disk

35 Metalo β-lactamase confirmation 35

36 What Labs Should Do Now Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC 2 mg/ml or nonsusceptible to ertapenem by disk diffusion Consider confirmation by Modified Hodge Test Can submit initial isolate for confirmation by bla KPC PC if KPC-producers not previously identified in hospital s isolate population Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate 36

37 KPC Questions If I have detect KPC-production, should I change susceptible carbapenem results to resistant? Not enough data to make a clear recommendation Clinical outcomes data will be necessary 37

38 THANK YOU 38