Limited utility of MALDI-TOF MS for identifying optrA-carrying vancomycin-resistant Enterococcus faecium during a hospital outbreak.

Enterococci are commensals of the human gut that opportunistically cause over 10% of hospital-acquired infections. Vancomycin-resistant enterococci (VRE) are often treated with linezolid; however, resistance to linezolid has emerged, most commonly mediated by the optrA gene. Bacterial strain typing is currently performed using whole-genome sequencing (WGS) at specialised laboratories. Matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is an established technology for microbiological species identification and has more recently been explored as a method for bacterial strain typing, with mixed results. MALDI-TOF MS has not previously been reported for the strain typing of optrA-carrying vancomycin-resistant Enterococcus faecium (optrA-VREfm). In this study, we examined the utility of MALDI-TOF MS to identify optrA-VREfm during a 2024 hospital outbreak at St Vincent's Hospital, Melbourne, Australia. Fourteen test isolates and 15 control isolates were included in this study. Isolates were characterised using the current gold standard WGS. MALDI-TOF MS spectral data from known outbreak and non-outbreak isolates were used to generate peak matrices, from which Euclidean distances were derived to generate dendrograms, and assessed for clustering of related strains. MALDI-TOF MS spectra were also visually inspected at mass regions identified by previous study to be useful in the strain typing of wild-type E. faecium, vanA VRE and vanB VRE. Using MALDI-TOF MS spectral data, the optrA-VREfm outbreak strains did not form a distinct cluster compared with control strains, when assessed against WGS. Visual inspection of the spectra at specific mass regions of interest also failed to reproduce results from a previous study to discriminate between any strains of E. faecium. These results do not support the use of MALDI-TOF MS for the typing of optrA-VREfm. Given the molecular properties of the optrA protein, MALDI-TOF MS may be intrinsically limited for identifying isolates carrying this resistance mechanism; however, further study is required to confirm this.