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MALDI-TOF MS and Resistance Detection
Beyond β-Lactamases
Jaroslav HrabákESCMID eLibrary
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Content of the presentation
• MALDI-TOF MS and its applications in microbiology
• MALDI-TOF MS for detection of antibiotic resistance
• Practical approach for development of new assays
• Conclusions
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Matrix-Assisted Laser Desorption/Ionization
Time-of-Flight Mass Spectrometry
(MALDI-TOF MS)
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MALDI-TOF MS Principle and Its Applications
in Microbiology
• Detection of different molecules (especially peptides and proteins) withprecise determination of molecular weight
• Since 2000th, MALDI-TOF MS has been applied for microbe identificationin routine diagnostic laboratories
• MALDI-TOF MS is a powerful diagnostic tool with many possibleapplications
LaserMatrixAnalyte
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MALDI-TOF MS for detection of antibiotic
resistance
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Need for Detection of Antibiotic Resistance
and its Mechanisms
• Rapid categorization of clinical isolate (bacteria, fungi)to susceptible/resistant category
• Detection of resistance mechanisms for:
• interpretative reading
• epidemiological purpose
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Rapid Categorization of Clinical Isolate to
Susceptible/Resistant Category
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Categorization of Clinical Isolate
General accepted parameters:• MIC• MBC (Minimum Bactericidal Concentration)• MAC (Minimum Antibacterial Concentration)
For MALDI-TOF MS:
• Minimum Profile Change Concentration (MPCC)
• Minimum Effective Concentration (MEC)
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Profile-Change Determination
• Detection of profile change after exposition to antibiotics/antimycotics
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Profile-Change Determination
• Validation of this technique for antifungal susceptibility testing
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Profile-Change Determination for Bacteria
• Cannot be done easily as in bacteria
• Media containing stable isotopes (e.g. 13C to 12C and/or 15N to 14N)
• Quantitative evaluation of protein expression
• Resulting in the shift of the peaks in spectra if using break-point concentration of the antibiotic
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Profile-Change Determination for Bacteria
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Detection of Bacterial Growth
• Proper internal standard allows semi-quantitative MALDI-TOF MS
• Detection of bacterial growth as in spectrophotometric assays
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Combination of Methods for Susceptibility
testing Using MALDI-TOF MS
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Prediction of Susceptibility by MALDI-TOF MS
• The response of bacteria to antibiotics is not a rapid change but specifickilling curves with more or less growth continuation are observeddepending on certain antibiotic (proposal of new parameters MPCC, MEC)
• Lower turnaround time comparing with classical cultivation techniques
• Possible integration of those techniques to automatic lines
• Further optimization and validation of those methods are needed
• Not so big advantage comparing with spectrophotometric assay
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Detection of Resistance Mechanism
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Detection of β-Lactamases
• Detection of small molecules (such as antibiotics)
• Detection of β-lactam ring hydrolysis (real visualisation of indicator molecule and its degradation products)
• Routinely used in some microbiological laboratories
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Detection of β-Lactamases
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Detection of β-Lactamases
• Modification for OXA-48 carbapenemases is available (NH4HCO3 in thebuffer)
• High sensitivity and specificity
• Needs experience for spectra interpretation, but it can be easily learned
• Not yet available as a commercial assay
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MRSA Detection
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MRSA Detection
• First study published in 2000 (Edwards-Jones et al.)
• Detection of “MRSA” specific peaks in mass spectra obtained from intactcells
– 14 detected in MRSA– 2 in MSSA
• MRSA/MSSA specific peaks using Surface-Enhanced LaserDesorption/Ionization Time-Of-Flight mass spectrometry (SELDI-TOF MS)(Shah et al. 2012)
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MRSA Detection – Clone-Specific Peaks
• Detection of Phenol Soluble Modulin (2415 m/z) encoded on SCCmeccassette I, III and VIII (Josten et al. 2014)
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Detection of Vancomycin—Resistant
Enterococcus spp.
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Detection of VRE
• First “successful” identification of vancomycin-resistant enterococci
• No isogenic strains were used for validation of the assay
• Detected peak are probably clone-specific
• There is currently no method for “universal”detection of VRE
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Detection of Porins
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Detection of Porins
• Use of sodium lauroyl sarcosinate for semi-specific extraction of cell-wall components
• Shorter turnaround-time compared with SDS-PAGE
• Fingerprintig of the proteins could help to precise identification of porins and its mutations
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Detection of β-Lactamases
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Detection of β-Lactamases
• Detection of TEM-1 β-lacatmase in isogenic strain (Camara and Hays,2007)
• Detection of CMY-2 and other β-lactamases in wild strains (Papagiannitsiset al. 2014)
– Specific extraction of periplasmic proteins
– MALDI-TOF MS detection of molecular weight
– Ability to detect acyl-enzyme complex
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Detection of β-Lactamases
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Conclusions
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Development of New Assays for Detection
of Antibiotic Resistance
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Conclusions
1. MALDI-TOF MS has many possible applications in diagnostic laboratories
2. Functional assays allow a detection of resistance mechanisms (currently β-lacatmases/carbapenemases)
3. Specific markers should be proposed for other resistant microbes (e.g., MRSA, VRE)
4. Precise validation of all assays is needed
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