MALDI-TOF MS and Resistance Detection Beyond ... - ESCMID

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FACULTY OF MEDICINE IN PILSENCHARLES UNIVERSITY IN PRAGUE

CHARLES

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IN PRAGUE

MALDI-TOF MS and Resistance Detection

Beyond β-Lactamases

Jaroslav HrabákESCMID eLibrary

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www.biomedic-plzen.cz FACULTY OF MEDICINE IN PILSENCHARLES UNIVERSITY IN PRAGUE

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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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• 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 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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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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Thank you for your attention

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MALDI-TOF MS and Resistance Detection Beyond ... - ESCMID

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