Rapid identification and resistance assessment: The future is mass spectrometry

Transcription

1 Rapid identification and resistance assessment: The future is mass spectrometry Dr Sanmarié Schlebusch Director of Microbiology Mater Pathology Brisbane

2 Outline Introduction Plug and play Pre-prep and post analysis Other wonderful approaches Other organisms Closing statement

3 Introduction MS is broad spectrum not restricted to pre-specified targets Rapid Masses of intact molecular ions and fragments easily determined by various types of MS Tandem MS can produce fragment ions which provide information about molecular structures and sequences Proteomics determines gene expression does not focus on only a single gene Protein expression may vary but gene detection may not represent protein expression at all

4 MALDI-TOF MS

5 MS and Tandem MS (MS/MS) MS MS/MS

6

7 Plug and play MRSA variable reports VRE routine use for VanB, fully automated Beta-lactamases B. fragilis cfia positive (MBL) vs cfia negative Ampicillin resistant E. coli poor reproducibility Clonal differentiation of resistant and susceptible clones cannot be expected for the majority of bacteria

8 Preparation and analysis required: Detection of enzymatic activity Beta-lactamases Variety difficult to propose universal primers MS detection of antimicrobial and degradation product Antimicrobial and organism+antimicrobial Spectrum represents beta-lactam molecule, its salts or degradation products is analyzed Detect loss of molecule peaks and presence of hydrolysis products after couple of hours Described for ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem and meropenem

9 Hydrolysis of beta-lactam ring corresponds to a mass shift of +18Da Ampicillin after (A) incubation with the β-lactamase-negative E. coli strain DH5α (B) a β-lactamase-producing strain (C) Inhibition of hydrolysis by a β-lactamase-producing strains was performed in the presence of clavulanic acid. Nonhydrolyzed form of ampicillin peaks are highlighted in gray. Hydrolyzed form of ampicillin peaks are indicated with a small black arrow.

10 Detection of enzymatic activity Carbapenem resistance Phenotypic and genotypic testing issues with TAT, cost, specificity MS rapid, easy, readily available reagents Enterobacteriaceae, P. aeruginosa and B. fragilis incubated with known MBLs and carbapenemases 67/70 carbapenemase producers correctly detected Imipenem and degradation products Large proportion OXA type carbapenemases in A.baumannii

11 Ertapenem degradation Ertapenem + NDM-1-carrying K. pneumoniae + IMP-1-carrying P. aeruginosa

12 Detection of enzymatic Carbapenem resistance activity Hydrolysis of ertapenem by K. pneumoniae directly from positive blood cultures Standardization of Acinetobacter carbapenemase detection (May 2013) Comparative analysis of MBL and OXA type producing Acinetobacter strains resulted in reduction of imipenem peaks Different effects was observed in different OXA type producers

13 MALDI-TOF MS spectra showing the most representative imipenem peaks (A) Incubation solution only (B) Imipenem-cilastatin solution (C) OXA-24 like producing Acinetobacter added (D) IMP-8 Acinetobacter added

14 Representative comparison of the average intensities of the most representative peaks of imipenem (m/z 300 and 489) obtained after a 1-h incubation with the different carbapenemases included in the study

15 Detection of enzymatic activity - conclusions All studies similar results this methodology has great potential to become routine Principle of degradation product monitoring can be applied to other enzymatic resistance mechanisms However, manual measurement and raw spectra analysis For ertapenem and K.pneumoniae in positive blood cultures MS calibrated to detect mass accuracy less than 0.5 Da Interpretation and evaluation expert knowledge required After peak assignment of sensitivity and resistance patterns classification becomes easy Special software and automation of acquisition and interpretation of results should be investigated and made available Not able to detect other carbapenem resistance mechanisms. These would include porin alterations and efflux mechanisms for K. pneumoniae, P. aeruginosa and porin alterations, efflux mechanisms and PBP alterations for A. baumannii.

16 Preparation and analysis required Other rrna methyltransferase detection Purified ribosomes and purified enzymes Needs to be simplified to become routine Porin detection OmpK36 porin in K. pneumoniae (responsible for penetration of carbapenems into periplasm) Failure to express OmpK36 due to an insertional inactivation or a nonsense mutation in the ompk36 gene Specific extraction followed by MALDI-TOF MS Porins detected on molecular weight

17 Carbapenem-resistant K.oxytoca ZC101; and K. pneumoniae Z5 and Z4. The arrows with dotted lines indicate the loss of 19,000- and 38,000-m/z peaks representing OmpK36.

18 Other approaches Proteomic approaches Entire protein complement expressed is studied Selective lysis and electrophoresis MALDI-TOF MS after SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) Described for outer membrane and periplasmic proteins altered in resistant isolates not routine

19 Other approaches Tandem MS Kanamycin phosphorylation detection Minisequencing DNA amplification, then allele-specific reactions measured by MALDI- TOF MS Specialized

20 Other approaches PCR-ESI MS Measures m/z of PCR amplicons Described for antimicrobial resistance detection Detection of mixed bacteria and antimicrobial resistance detection eg. meca or vana/b (specific primers used) in IE Nanoparticles MALDI-TOF MS not suitable for detection of small molecules due to matrix interference Nanoparticles for example magnetic nanoparticles can enrich and enhance detection of small molecules Improves signal to noise ratio

21 Other organisms Antifungal susceptibility C. albicans and fluconazole Proteome monitored of Candida cells grown with and without antifungal C. albicans and caspofungin CCI on standard Bruker Microflex used Antiviral susceptibility RFMP assay based on MS analysis of small DNA fragments that include sites of mutation Distinct peaks relevant to each codon NRTIs, NNRTIs and PIs for HIV drug-resistance Fast, cost effective Bruker Biflex

22 Summary MALDI-TOF MS Great potential Direct detection of antimicrobial resistance (plug and play) already in use Enzyme degradation of antimicrobials already in routine use Develop more methods Better software and automation Other approaches Being investigated Other resistance mechanisms Other MS and technology Different organisms

23 Final statement Proteome-level studies detect behavior of strains Expression of proteins of interest Posttranslational modifications Most routinely used molecular genetics are restricted to detection of known resistance determinants Including WGS databases Some proteomic analysis labor-intensive reference centers MALDI-TOF MS opens new avenues for clinical and experimental microbiology

24 Then NOW Daniel Zeevi Apr 14, 2013

25 References Hrabák et al. Clin. Microbiol. Rev. 2013;26 (1): Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry for Detection of Antibiotic Resistance Mechanisms: from Research to Routine Diagnosis Demirev et al. Annu. Rev. Anal. Chem : Mass Spectrometry for Rapid Characterization of Microorganisms Holland et al. Anal. Chem. 1999;71(15): Identification of Bacterial Proteins Observed in MALDI TOF Mass Spectra from Whole Cells Carbonelle et al. Clin Biochem. 2011;44(1): MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Shah et al. Clin. Microbiol. 2012;50(3): Tracing the transition of methicillin resistance in sub-populations of Staphylococcus aureus, using SELDI-TOF Mass Spectrometry and Artificial Neural Network Analysis. Griffin et al. J Clin Microbiol. 2012;50(9): Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak. Kok et al. Pathology. 2013;45(1):4-17. Current status of matrix-assisted laser desorption ionisation-time of flight mass spectrometry in the clinical microbiology laboratory.

26 References Sparbier et al. Clin. Microbiol. 2012;50(3): Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry-Based Functional Assay for Rapid Detection of Resistance against β-lactam Antibiotics Burckhardt et al. J Clin Microbiol. 2011;49(9): Using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry To Detect Carbapenem Resistance within 1 to 2.5 Hours. Álvarez-Buylla et al. J Clin Microbiol. 2013;51(5): Optimized method for Acinetobacter species carbapenemase detection and identification by matrix-assisted laser desorption ionizationtime of flight mass spectrometry. Smith et al. Anal Chim Acta. 2013;786: A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase. Lee et al. Clin Microbiol Infect. 2013;19(6):E Restriction fragment mass polymorphism (RFMP) analysis based on MALDI-TOF mass spectrometry for detecting antiretroviral resistance in HIV-1 infected patients. Vella et al. J Clin Microbiol. 2013; [Epub ahead of print]. Rapid Antifungal Susceptibility Testing by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Analysis. Kirpekar et al. RNA. 2000;6: Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry Lin et al. Anal. Chem. 2007;79: Functionalized Magnetic Nanoparticles for Small- Molecule Isolation, Identification, and Quantification.