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1 Conventional vs newly developed techniques for identification of resistance Laurent POIREL Medical and Molecular Microbiology Unit Dept of Medicine University of Fribourg Switzerland

2 ANTIMICROBIAL AGENT MICRO- ORGANISM PATIENT

3 Antibiogram Reading Ø Inh. zone (mm) MIC (mg/l) S-I-R

4 Clinical Categories CLSI (NCCLS): Clinical and Laboratory Standards Institute EUCAST: European Committee on Antimicrobial Susceptibility Testing SFM: Société Française de Microbiologie BSAC: British Society for Antimicrobial Chemotherapy MENSURA: Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos

5 Interpretative reading of the antibiogram 1.- Define phenotype of susceptibility and resistance 2.- Deduce the possible mechanism of resistance 3.- Adequate clinical categories to the inferred mechanism of resistance, and change phenotype if necessary

6 Interpretative reading of the antibiogram S-I-R Microbiological knowledge CLINICAL REPORT OF RESULTS

7 S-I-R AES SOFTWARE (Expert system) CLINICAL REPORT OF RESULTS

8 INTERPRETATION

9 Identification of the micro-organism (at the species level) Analysis of susceptibility/resistance phenotype - Groups (families) of antimicrobial agents - Antimicrobial indicators of resistance Define Phenotype Interpreative reading - Common phenotypes - Unusual phenotypes - Impossible phenotypes Deduce biochemical mechanism of resistance Clinical relevance of the inferred resistance Re-define clinical categories

10 E. coli Wild-type phenotype (ß-lactams) MOX CAZ ATM FEP AMC IPM CF CTT CXM AMX TIC PIP TCC CTX FOX TZP

11 E. coli Acquired penicillinase or K. pneumoniae MOX CAZ ATM FEP AMC IPM CF CTT CXM? AMX TIC PIP TCC CTX FOX TZP

12 E. coli High-level penicillinase MOX CAZ ATM TCC FEP AMC IPM CTX CF CTT CXM FOX AMX TIC PIP TZP

13 Requirements of interpretative reading of the antibiogram Identification of the microorganism Analysis of S/I/R phenotype Use of indicator agents Study antibiotic-inhibitor combinations Quantitative study of susceptibility Use of high inocula (in some cases) Local epidemiology information Availability of reference methods

14 Microorganism Clinical Relevance of the mechanism of resistance Identification + antibiogram Deduce Phenotype of Resistance Deduce Biochemical Mechanism of Resistance Re-Define Clinical Categories, if necessary Deduce susceptibility/resistance to non tested antimicrobial agents Antibiogram Interpretation REPORT

15 Livermore et al., JAC 2001

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20 Limitations to interpretative reading of the antibiogram High complexity of resistance mechanisms Limited information about some mechanisms of resistance Low level resistance Multifactorial multiresistance Oversimplification of interpretative reading Mistakes when deducing mechanisms of resistance

21 Benefits of the interpretative reading of the antibiogram Adequacy of antimicrobial therapy Detection of new mechanisms of resistence Analysis of epidemiology of resistance Antimicrobial policy Improved quality in laboratory testing

22 How to detect carbapenemases?

23 Question ; any carbapenemase here? K. pneumoniae K. pneumoniae K. pneumoniae E. coli IMP ETP E. coli IMP MEM MEM MEM ETP E. coli ETP ETP IMP ETP ETP IMP IMP IMP MEM MEM MEM

24 The reasons of the complexity Nordmann et al. Clin Microbiol Infect 2012; 18:432-38

25 Susceptibility testing : imipenem, ertapenem, meropenem: CLSI, EUCAST guidelines Phenotypic detection - Hodge test; modified Hodge test - Inhibition; EDTA, clavulanic acid, boronic acid Carbapenem hydrolysis (UV spectrophotometry, Mass spectro) Molecular biology Detection of carbapenemase producers in infected samples - Specific PCR, multiplex PCR +/- sequencing - Real time PCR - DNA Microarray

26 D 0 D 1 D 2 Urine Infections Other samples + Antibiogram Blood cultures + Antibiogram

27 When to suspect production of a carbapenemase? Clinical breakpoints and screening cut-off values for carbapenemase-producing Enterobacteriaceae

28 Principal - KPC inhibited by boronic acid or clavulanic acid - MBL inhibited by EDTA or dipicolinic acid Tests available Inhibition tests - Combined Test (ROSCO) : meropenem +/- cloxacillin or dipicolinic acid or boronic acid - E-test MBL - Inhibition by EDTA («home-made» technique) Imipenem + EDTA Imipenem alone

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30 Modified Hodge test Strain to be tested Positive control Negative control

31 - Low sensitivity with no specificity for different carbapenemases - Variable results with different carbapenems - False negative results: - weak results with certain MBLs (NDM) (increased sensitivity with ZnSO 4 ) - False positive results: - CTX-M-15 or AmpC hyperproduction CTX-M-15 Modified Hodge test CTX-M-15 C(+) AmpC AmpC C(+) CTX-M-15 C( )

32 T- Value of the Modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae OXA-48 NDM-1 (a) MH Girlich D, Poirel L, Nordmann P, J Clin Microbiol KPC-2 NDM-1 (b) NDM-1 (c) T- OXA-48 NDM-1 (a) KPC-2 NDM-1 (b) MH + ZnSO4 (100 µg/ml) NDM-1 ( c )

33 Disk diffusion synergy test: IMP + EDTA or CAZ + EDTA CAZ EDTA IMP

34 IMP IMP + EDTA Combined-disk tests AMC ATM

35 KPC: synergy with boronic acid IMP IMP + boronicac. boronic ac. Disk diffusion synergy test: IMP + boronic ac. Disk combination test: carbapenem + boronic ac.

36 Another basic and useful method (for expert labs) Measurement of carbapenem hydrolysis by UV spectrophotometry - 10 µl of bacterial crude extract µm of imipenem - wavelength: 297 nm Bernabeu, Poirel & Nordmann, DMID 2012

37 Protocol : Mass spectrometry : MALDI-TOF 1) Broth culture with the strain to be tested + carbapenem : 3-6h 2) Mass spectrometry 3) if carbapenemase + : hydrolysis of the carbapenem Carbapenem molecule leading to a hydrolysis product degradation product Advantages : Specific / sensitive Fastness + Cheap if you dispose from the machine! Disadvantages Material price Expertise Carbapenem NDM-1 IMP-1 Hrabák et al. JCM Burckhardt et al. JCM Hrabák et al. JCM. 2012

38 Molecular biology : PCR-based Techniques Real-Time PCR : - Check-MDR Real-Time PCR - Detect the presence of the carbapenemase gene h - Cost +++ Specific PCR +/- sequencing : - OXA-48-like / KPC / VIM / IMP / NDM - 3 to 5 h - Expertise ++ - Cost +

39 Molecular approaches - simple and multiplex PCR + sequencing 3 multiplex reactions: #1: blaimp, blavim, blaspm #2: blandm, blakpc, blabic, blaoxa-48 #3: blaaim, blagim, blasim, bladim Poirel et al. Diagn Microbiol Infect Dis 2011; 70: real time PCR for non-mbl: blages, blaimi/nmc, blakpc, blaoxa-48, blasme Swayne et al. Int J Antimicrob Chemother 2011; 38:35-8

40 PCR + MASS SPECTROMETRY PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) Main application for identification of micro-organisms Multiplexing Desalting Analysis by mass-spec Possible for resistance mechanisms : -meca, mupa, erma, ermc -vana et vanb -bla KPC -INH et RIF (MTB) -FQ (gyra, parc) Ecker et al., Nat Rev Microbiol 2008;6:553-6 Lavigne et al., Clin Chem Lab Med 2012;0:1-14

41 Molecular tests: commercial microarrays - colony: sensitivity and specificity of 100% for KPC, OXA-48 and MBLs - clinical samples: not yet evaluated Naas et al. J Clin Microbiol 2011; 49: Willemsen et al. J Clin Microbiol 2011; 49: Bogaerts et al. Antimicrob Agents Chemother 2011; 55:

42 Next step automation, multiplexation

43 No Specialized Training Required to Achieve Reliable, Reproducible Results INTEGRATED PLATFORM AND TEST 1 Insert Swab into Elution Reagent Vial and Break at Score 2 Vortex and Dispense Sample into Port S Total Hands-On time <1 Minute 3 Insert Cartridge and Start Assay

44 Xpert MDRO Cartridge Cartridge detects three carbapenem resistance gene families (54 genes in total) bla KPC bla NDM bla VIM Sample : Rectal Swabs Result in 47 minutes

45 Updated Targeted Xpert Test Menu U S In tl 14 Tests MRSA Surveillance SA Nasal Complete C. diff & C. diff/epi vana for VRE MRSA/SA SSTI MRSA/SA Blood Culture CT/NG EV Flu GBS & GBS Lim Broth Factor II&V MTB/RIF 16 Tests MRSA Surveillance SA Nasal Complete C. diff/epi vana/vanb VRE MRSA/SA SSTI MRSA Blood Culture EV Flu GBS Factor II&V BCR/ABL v1 & BCR/ABL v2* CT & CT/NG HPV* MTB/RIF 18 Tests Trichomonas Norovirus Flu/RSV BCR/ABL v Tests 36 Tests Tests Norovirus CARBA-R (MDRO) Trichomonas CT/NG LBC HIV Viral Load HIV Qualitative HCV Viral Load Flu/RSV Bladder Monitor/Sympto HSV 1/2 Typing CT/NG LBC Vaginitis CARBA-R (MDRO) HIV Viral Load HCV Viral Load Bladder Monitor Bladder Symptomatic CW Flu/RSV HBV Viral Load HSV 1/2 Typing Vaginitis Group A Strep Sepsis Fungal Respiratory Panel Gastro Panel OncoScreen Lung Prostate Recurrence Risk Breast Recurrence Sig Breast Therapy Stratifier 42 Tests HPV CW CT/NG HBV Viral Load CW Vag Group A Strep CW GBS Sepsis Fungal CW GAS Meningitis/Encephalitis Respiratory Panel Gastro Panel OncoScreen Lung Prostate Recurrence Risk Breast Recurrence Sig Breast Therapy Stratifier Tests Meningitis/Encephalitis Targeted Test Menu Subject to Revisio

46 R O N COOH Carbapenems Imipenem Meropenem Ertapenem Doripenem S-R The Carba NP test Carbapenemase Colorimetric detection HO O R H 2 N COOH S-R Acid production ph

47 Lysis buffer The Carba NP test; the kit 3 mg of imipenem powder Diluted red phenol ph=7.8 + ZnSO mm 96 wells plate

48 K. pneumoniae CTX-M-15 + impermeability K. pneumoniae OXA-48 K. pneumoniae KPC-2 E. coli VIM-1 Results E. coli IMP-1 Carbapenem + - Yellow = carbapenem hydrolysis E. coli NDM-1

49 Ambler class The Carba NP test Carbapenemase type Mean time for positivity A KPC 15 min- 1h A GES-2, -5 1h-1h30 B NDM min B VIM min B IMP 5-30 min D OXA min

50 Question ; any carbapenemase here? K. pneumoniae K. pneumoniae K. pneumoniae E. coli IMP ETP E. coli IMP MEM MEM MEM ETP E. coli ETP ETP IMP ETP ETP IMP IMP IMP MEM MEM MEM

51 E. coli VIM-1 Question : any carbapenemase here? K. pneumoniae CTX-M15 + impermeability K. pneumoniae OXA-48 K. pneumoniae KPC-2 (-) 2 hours (+) 30 min (+) 5 min E. coli IMP-1 E. coli NDM-1 (+) 30 min (+)25 min (+) 20 min

52 The Carba NP test 1- Rapid; less than 2 h 2- Sensitive; 98% (1,600 tested strains, French National Reference Center) 3- Specific: 100% 4- Detection of any type carbapenemase activity 5- Cheap : 0.5 euro 6- Easy-to-handle 7- Implementable worldwide

53 This test is now commercially available

54 How to detect extended-spectrum ß-lactamases?

55 ESBL Detection on a disc diffusion antibiogram

56 Principal - ESBLs inhibited by clavulanic acid or tazobactam - Double-disk synergy test Inhibition tests - E-test CAZ / CAZ + clav., or FEP / FEP + clav.

57 R N O S COOH Cefotaxime R Detection of ESBLs : ESBL NDP test ESBL + tazobactam HO Production of acid O R S R S N R O COOH H 2 N R COOH ph Nordmann, Dortet, Poirel J. Clin. Microbiol.

58 D 0 D 1 D 2 Urine Infections Other samples + Antibiogram Blood cultures + Antibiogram ESBL NDP test Carriage = stools

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61 High-throughput sequencing Theoritically, will allow : - accurate identification - obtention of a «virtual» antibiogram - typing (criteria to be defined) Preparation of the sample is critical Gene expression level Cost Dune et al., Eur J Clin Microbiol Infect Dis 2012;31:

62 Sequencers of the 3 rd generation Quail et al., BMC Genomics 2012;13:341

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65 JAC, 2012

66 TRANSCRIPTOME BY RNA-SEQ Robust and reproducible method HOWEVER Sample preparation is critical and difficult Quite long process bio-informatical analysis Costly Febrer et al., Trends Biotechnol

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73 Conclusion - Increased prevalence of carbapenemase producers worldwide. - Very few novel antibiotics will be launched within the next years. - Diagnostic techniques are now available for accurate diagnostic of carbapenemase producers - Two main goals: - - Carbapenem stewarship - - Outbreak prevention and control

74 Phenotypic Inhibition of bacterial growth Current reference May be fastened (micro-fluidic, nanotechnologies) Fastness/Reliability? CONCLUSION Methods Genotypic Direct detection of resistance Multiplexable Bio-informatic Hetero-resistance? Unknown mechanisms? Complementary approaches mandatory

75 Thank you