Affiliations: School of Allied Health Sciences, De Montfort University, Leicester, UK | Research Institute of Hygiene, Occupational Pathology and Human Ecology, St. Petersburg, Russia | Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, St. Petersburg, Russia | Clinical Microbiology, University Hospitals of Leicester, Leicester, UK
Note:  Corresponding author: Richard O. Jenkins, School of Allied Health Sciences, De Montfort University, Leicester, LE1 9BH, UK. Tel.: +44 116 2577942; E-mail: firstname.lastname@example.org
Abstract: BACKGROUND: Limited success has been reported for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) differentiation of staphylococci, including methicillin resistant Staphylococcus aureus (MRSA) strains. Chemical pretreatment of cells prior to MALDI-TOF-MS analysis has not been systematically investigated for enhanced discrimination of S. aureus strains. OBJECTIVES: To evaluate various chemical pretreatment of cells for MALDI-TOF-MS discrimination of clinical staphylococcal isolates, with a focus on differentiation of MRSA from methicillin sensitive S. aureus (MSSA) strains and from other staphylococcal species. METHOD: MALDI-TOF-MS of a well-characterised S. aureus strain(s) was optimised with respect to matrix chemical(s), matrix solvent and target plating method. Various chemical pretreatments (solvents, reductants, detergents) and pretreatment application methods were then evaluated for enhancement of spectral richness. The three most promising pretreatments were applied to MALDI-TOF-MS discrimination of three set of clinical isolates comprising non-S. aureus staphylococci (77 isolates), MSSA (36) and MRSA (43), with analysis by total or set specific resolved peaks. RESULTS: The optimized MALDI-TOF-MS protocol involved α-cyano-4-hydroxycinnamic acid (CHCA) as matrix chemical (in 1:2 acetonitrile:H2O and 2% trifluoroacetic acid), with application as an overlay onto smeared cells (on-probe). On-probe application of chemical pretreatment was most effective at enhancing MALDI-TOF-MS spectral richness. Use of reductants and detergents as pretreatments were ineffective. The three most effective solvents/acid pretreatments – ethanol:formate, ethanol:acetate and formate:isopropanol – each generated reproducible and distinct spectra over the 2,000–10,000 m/z range. For the combined sets of clinical isolates (114), all three of these pretreatments increased the total number of resolved peaks in comparison with no pretreatment controls. The ethanol:formate pretreatment gave 100% clustering of non-S. aureus staphylococci, based on total resolved peaks. The formate:isopropanol pretreatment generated the largest increase in number of MRSA set specific peaks (from 18 to 32; 78% increase) and clustered the majority (77%) of the MRSA strains together, although compete discrimination of the MSSA and MRSA was not achieved. CONCLUSION: MALDI-TOF-MS discrimination of clinical isolates of staphylococci is enhanced through chemical pretreatment of cells. Three chemical pretreatments, not previously applied to staphylococci, are highlighted for enhancing spectral richness and offering new opportunities for improved discrimination of staphylococci, including MRSA and MSSA strains.
Keywords: MALDI-TOF-MS, Staphylococcus, S. aureus, MRSA, MSSA