Liquid extraction surface analysis mass spectrometry of microbes: towards point-of-care diagnosis for wound infection

Havlikova, Jana ORCID: 0000-0002-7557-4733 (2022). Liquid extraction surface analysis mass spectrometry of microbes: towards point-of-care diagnosis for wound infection. University of Birmingham. Ph.D.

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Abstract

Trauma is one of the leading causes of death of people under age 49, while complications associated with the wound infection are the primary cause of death in the first day of injury. Microbial infections are mostly caused by bacteria and fungi commonly present in the hospital environment and these are associated with antibiotic and antifungal resistance. Rapid and accurate microbial identification is therefore of high importance. Current diagnostics takes hours or even days to obtain the result. This work uses liquid extraction surface analysis mass spectrometry (LESA MS) for top-down (TD) analysis of proteins in clinically important microbes including the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) and yeast Candida glabrata. First, the ESKAPE microbes and their two close clinical isolates were grown on simple substrates and subjected to LESA MS. Searches against individual protein databases resulted in identification of 24 proteins from 37 MS/MS mass spectra, while searching against multiple databases determined identification success rate at protein and species level to be 79%. A biofilm model of P. aeruginosa and C. glabrata was cultured, resulting in identification of three P. aeruginosa proteins. Next, a LESA MS workflow was developed for analysis of wounded in vitro three-dimensional living skin models inoculated with four of the ESKAPE species and C. glabrata, resulting in assignment of seven human, nine bacterial and one yeast proteins. The LESA MS workflow was subsequently applied to ex vivo human skin grafts including those inoculated with S. aureus, and two human skin proteins were identified from both types of samples. Lastly, LESA MS was coupled to state-of-art cylindrical high-field asymmetric waveform ion mobility spectrometry (FAIMS) and mass spectrometer and a high-throughput approach for TD identification of proteins in bacteria was developed. In total, the high-throughput TD LESA FAIMS analysis of Escherichia coli K12 and four ESKAPE species allowed identification of 179 proteins and 277 proteoforms. The results presented in this thesis suggest that LESA MS and LESA FAIMS MS are tools capable of relatively fast extraction of proteins from microbes cultured on a variety of substrates with their subsequent tandem MS identification, showing potential of LESA as a future in situ diagnostic.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):