MSACL 2018 US Abstract

Topic: Microbiology/Virology

Metal Oxide Laser Ionization MS Bacterial Fatty Acid Composition for Rapid ID and Antibiotic Resistance Determination

Chris Cox (Presenter)
Colorado School of Mines

Bio: I received my B.S. in Microbiology and Biochemistry from the University of Oklahoma. I then studied under Dr. Michael Gilmore, first at the OU Med. School to obtain a Master’s in Microbiology, then at Harvard where I did my dissertation research and completed my Ph.D. I came to Mines as a postdoc to investigate the use of phages and various forms of mass spectrometry for molecular biomarker measurement and microorganism ID. Now, as a junior member of the research faculty as CSM, my research focuses on development of new bacterial and fungal diagnostics. By focusing on phage amplification or bacterial and fungal fatty acid measurement by LFI, MALDI or MOLI-MS, Raman spectrometry and lateral flow capillary concentration (LFCC) (a novel technology that I invented), we have developed more accurate, user-friendly diagnostic approaches with broad commercialization potential.

Authorship: C.R. Cox1*, K.R. Jensen2, N.R., Saichek1, P.B. Harrington3 and K.J. Voorhees1
(1)Advanced Biodetection Technology Laboratory Department of Chemistry Colorado School of Mines, Golden, CO U.S.A. (2)Osaka University, Osaka Japan. (3)Ohio University, Athens, Ohio

Short Abstract

MALDI-TOF MS protein profiling has emerged as a rapid approach for clinical bacterial diagnostics. However, current methods do not address the increasing demand for antibiotic resistance profiling and often cannot accurately ID closely related phylotypes. As a result, additional culture-based tests are required to inform antibiotic therapy and confirm ID if protein-based methods fail. This adds significant time and expense, further delaying patient treatment and reducing the likelihood of positive therapeutic outcomes. We investigated metal oxide laser ionization (MOLI) MS fatty acid analysis as an alternative and achieved highly accurate simultaneous strain-level ID and resistance profiling of methicillin resistant Staphylococcus aureus and aminoglycoside, cephalosporin and fluoroquinolone resistant Pseudomonas aeruginosa.

Long Abstract

Introduction

MALDI-TOF MS protein analysis has emerged as a rapid approach for clinical bacterial diagnostics. However, current protein-based methods do not address the increasing demand for antibiotic resistance profiling and they often cannot differentiate closely related phylotypes that in many cases express similar if not identical proteins. This can result in misidentification or failure to provide any reliable identification at all. As a result, additional culture-based testing is typically required to better inform antibiotic therapy. This adds significant time and expense, further delaying patient treatment and reducing the likelihood of positive therapeutic outcomes.

 

Methods

New approaches that combine accurate ID and resistance profiling into a single rapid test would drastically reduce turnaround and improve treatment of drug resistant infections. To address this, we investigated metal oxide laser ionization mass spectrometry (MOLI MS) fatty acid analysis as a means of simultaneous strain-level ID and antibiotic resistance profiling. By focusing on bacterial lipids as diagnostic biomarkers rather than proteins, and by exploiting the unusual in situ catalytic propensity of the rare-earth lanthanide CeO2 to cleave taxonomically viable fatty acids from bacterial lipids, we obtained highly accurate and reproducible species- and strain-specific bacterial profiles using conventional MALDI instruments already widely in clinical use.

Results

MOLI MS fatty acid profiling resulted in reproducible classification at the species and strain level as determined by principal component analysis and statistically validated by leave–one-out cross validation. Notably, this approach simultaneously allowed for 100% accurate classification and differentiation of a diverse collection of Gram positive methicillin resistant and susceptible Staphylococcus aureus (MRSA/MSSA) isolates. Similarly, 98%, 96% and 90% correct classification of ceftazidime, ciprofloxacin and gentamicin resistant Gram negative P. aeruginosa was observed.

Conclusions & Discussion

Novel CeO2-catalyzed MALDI-TOF MS provides a more accurate alternative to protein-based methods. By coupling the catalytic activity of CeO2 with MALDI laser energy, we demonstrated in situ decomposition of bacterial lipids into taxonomically useful fatty acid constituents.  This allowed for highly accurate ID and differentiation of MRSA and aminoglycoside, cephalosporin, and fluoroquinolone resistant P. aeruginosa. MRSA and MSSA were differentiated with 100% strain-level accuracy. Resistant and susceptible P. aeruginosa were differentiated with 90-98% strain-level accuracy. The primary advantages of this new diagnostic technique are the avoidance of misidentification of closely related phylotypes, greatly improved accuracy, and simultaneous ID and determination of antimicrobial resistance in a single rapid test, which can improve therapeutic management and infection control.


References & Acknowledgements:


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