Screening for Resistant Organisms and Infection Control

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1 Screening for Resistant Organisms and Infection Control Dr Sonal Saxena Professor Department of Microbiology Lady Hardinge Medical College New Delhi 1

2 MDRO The proportion of K. pneumoniae and E. coli with resistance to fluoroquinolones, thirdgeneration cephalosporins, aminoglycosides and a combined resistance to all three antibiotic groups has increased significantly. Similar patterns are observed with Acinetobacter spp., and MRSA with resistance levels varying from 0.9 to 56 % depending on the country studied Fresno 11/ 2

3 ESKAPE pathogens Account for more than 80 % of infectious episodes in the ICU E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp. Fresno 11/ 3

4 MDROs have been divided into three categories depending on their resistance profile: 1. MDR non-susceptible to at least 1 agent in 3 antimicrobial categories 2. XDR non-susceptible to at least one agent in all but 2 or fewer antimicrobial categories 3. PDR non-susceptible to all agents in all antimicrobial Clin Microbiol Infect. 2012;18: categories

5 Screening for MDROs with infection control perspective Carbapenamase detection methods MHT CarbaNP mcim ESBL and AmpC detection Polymyxin detection MRSA Inducible clindamycin resistant VRE HLGR 5

6 ESBL testing Double disc diffusion MIC Chrome agar Expert systems Molecular tests 6

7 DDST M100S 27th ed. 7

8 Reading DDST 8

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10 Confusion galore DDST fails to detect ESBLs in the presence of AmpC, as the latter enzyme is resistant to CA. CA may induce high-level expression of chromosomal AmpC, masking the synergy arising from the inhibition of an ESBL. Boronic acid derivatives were reported as reversible inhibitors of AmpC enzymes. The usage of BA in combination with CA for the detection of ESBLs among AmpC-producing organisms. Use of cefepime with CA for the reliable detection of ESBL in the presence of AmpC enzymes. 10

11 Disk layout pattern (a); pure ESBL producing isolate (b); pure AmpC-producing isolate (c); ESBL and AmpC-producing isolate (d); inducible AmpC-producing isolate showing the inhibition of cephalosporin blunting after the addition of BA (e); isolate without harbouring any type of enzymes (f). 11

12 A 5 mm increase in the zone diameter of the FOX alone and in combination with BA was considered positive for AmpC production 12

13 CARBAPENEM RESISTANCE 13

14 Mechanisms at work Production of carbapenemase Outer membrane permeability Efflux pumps Synergistic activity between Amp-C and ESBL 14

15 Carbapenemases 15

16 Carbapenemase testing Modified Hodge test* Carba NP test mcim test* Chrome agar Expert systems * Only for Molecular test Enterobacteriacae 16

17 MHT Lawn of E.coli ATCC :10 dilution of 0.5 MacFarland 17

18 Modified Hodge Test Neg Control - KPC + NDM False - OXA False positive with ESBL/AmpC with porin loss Only applies to Enterobacteriacae 18

19 CARBA NP test Carbapenem hydrolysis by Carbapenemase producing bacteria acidifies the medium which results in the change in color of the ph indicator from red to yellow Detects in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumann: Class A: KPC is the main representative Class B: Metallo ß-Lactamases (MBL). NDM-1, VIM and IMP Class D: OXA Carbapenemases 19

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21 RAPIDEC CARBA NP Detects all 3 types of produced Carbapenemase: Class A: KPC is the main representative Class B: Metallo ßLactamases (MBL). NDM-1, VIM and IMP Class D: OXA Carbapenemases 21

22 Carba NP Method Apply to Enterobacteriacae, Pseudomonas and Acinetobacter spp. False Negatives may be due to Weak carbapenemases (e.g., OXA-48-like, SME-1, or GES-5) Source plate medium used for inoculum Time to positive may vary by type KPC quick Others slower (OXA slowest) Dortet et al Antimicrob Agents Chemother. 58:1269. Lee et al J Clin Microbiol. 52:4023. Tijet et al Antimicrob Agents Chemother. 58:

23 mcim 23

24 Only for Enterobacteriacae 24

25 Carbapenemase testing 25

26 Efflux pump Carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) acts as efflux pump inhibitors Agar dilution MICs of imipenem, meropenem and ertapenem with 50 and 100 mg/ml and without CCCP. The influence of an efflux pump on the carbapenems s MIC for a given bacterial strain is determined by a reduction of at least fourfold of the respective MIC in the presence of CCCP 26

27 Molecular testing Carbapenemase genes Efflux pump gene acrart in Enterobacter for OMP genes 27

28 Colistin testing 28

29 Problems! Poor diffusion in agar due to large molecule Polymyxins adsorb to polystyrene present in plates. This effect may be mitigated by the addition of a surfactant such as polysorbate80; which may act synergistically with the polymyxins and artificially lower minimum inhibitory concentrations. 29

30 EUCAST Antimicrobial susceptibility testing of colistin problems detected with several commercially available products. This warning was issued July 2016 and modified and extended 26 August 2016 and 20 June, The broth microdilution tests seem to give correct results both for susceptible and non-susceptible isolates. Disk diffusion cannot be used for susceptibility testing of colistin. It does not discriminate between susceptible and resistant isolates. Currently available gradient tests underestimate colistin MIC values and undercall resistance, and should be avoided, even when quality control results are within range. 30

31 EUCAST MIC Breakpoint tables 31

32 CLSI CLSI MIC breakpoints applicable to A. baumanii complex, Pseudomonas aeruginosa For Enterobacteriacae only ECV available in CLSI 32

33 GRAM POSITIVE ORGANISMS 33

34 MRSA MRSA phenotype can be highly heterogeneous. The accuracy detection methods is affected by inoculum size, incubation time and temperature, medium, ph, salt concentration and other factors. In addition, these detection methods require 24h incubation for accurate results. Cefoxitin disk screen test:<22mm: resistant MIC: 4 µg/ml 34

35 Enrichment broth containing cefoxitin and magnetic microparticle extraction of S. aureus followed by detection of adenylate kinase. Useful for nasal swab screeing. Mannnitol Salt Agar with oxacillin. - MRSA Screen Plate (Mueller Hinton Agar with 4% NaCl and 6µg/mL oxacillin). Chromogenic culture media. Latex particles sensitized with a monoclonal antibody against PBP2/ specifically react with MRSA to cause agglutination visible to the unaided eye. PBP2 Latex test 35

36 Vancomycin screen agar BHI agar with 6µg/ml vancomycin. Spot inoculate from 0.5 Mcfarland suspension. Control:E.faecalis ATCC Incubate 24 hours. >1 colony: S aureus : presumptive reduced susceptibilitydo MIC Enterococcus :Presumptive VRE. Do speciation for intrinsic Resistance then MIC. 36

37 Inducible Clindmycin Resistance 37

38 Chromogenic culture medium Combine early screening and detection of resistant phenotype. 38

39 Man over Machines Expert systems Detection of genes by molecular techniques 39

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42 Detection of enzymatic activity Carbapenemases: incubate bacteria and carbapenem, analyse the products with Malditof. carbapenemase if present can lyse the carbapenem then the specific spectra for the degradation products of the carbapenem can be detected. rrnamethyltransferase activity: Direct detection of enzymatic modification. (aminoglycosides, chloramphenicol & clindamycin) caused by methylation of 16s rrna. Others: Similar methods are also being developed to look for other beta-lactamases. 42

43 Direct analysis of bacterial extracts MRSA: Specific spectral peaks for MRSA are identifiable, but are quite subtle and difficult to reproduce in a consistent manner at present. Detection of VRE: Detection of the Van A and Van B genotypes now well validated in a research setting. 43

44 Minisequencing Involves using the fact that specific mutations produce specific differences in molecular mass. However it require DNA amplification and still quite labour and time intensive. Detection of ESBLs : Difficult This technique could also be used to detect mutations responsible for resistance in Neisseria gonorrhoeae, Streptococcus pneumoniae, H pylori, etc. 44

45 Proteomics Qnr Proteins: proteomic studies on multi resistant bacteria that allow the construction of a complete database of protein fingerprints of resistant isolates for the detection of the main proteins responsible for resistance. 45

46 New technologies Cepheid Xpert Carba-R assay: designed for the rapid detection and differentiation of the blakpc, blandm, blavim, blaoxa-48 and blaimp-1 genes. Sensitivity, specificity, positive and negative predictive values compared to reference culture and sequencing results were above 95 %. qmrsa: Quick, multiplex immunocapturecoupled PCR, able to decrease median time to notification from four days to one day. 46

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