The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria

Abstract

With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48 h of incubation for gram-negative anaerobes and after 72 h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24 h and 48 h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48 h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner.

Application of Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry for Identification of Coagulase-negative Staphylococci Isolated from Milk of Cows with Subclinical Mastitis

“…The concentration of cells observed has also been shown to influence the MALDI-TOF MS identification (Clark et al 2013, Veloo et al 2014. Applying approximately 1 × 10 7 bacterial cells from the 70% formic acid extraction onto the target plate resulted in better spectral resolution.…”

A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS

“…Among them are; the growth phase of the bacterium, the amount of bacterial cells spotted on the target, the type of colony, whether an extraction method is used and, for anaerobic bacteria, the time the cells are exposed to oxygen. Since the basis of the method is 'pattern matching of main spectral profiles (MSPs)', the inclusion of a reference spectrum in the database is necessary for successful identification [5,7,8,12]. Table 4 Performance of the laboratories for each strain.…”

“…Table 4 Performance of the laboratories for each strain. The fact that none of the participating laboratories was able to obtain a good spectrum of C. ureolyticus, emphasizes again that the nature of colony plays an important role for successful identification [12]. C. ureolyticus forms tiny pitting colonies which are very difficult to spot.…”

“…MALDI-TOF MS analyses were performed as described previously [12]. Briefly, for the direct spotting method, bacterial cells were transferred to a stainless-steel target using a toothpick.…”

“…Since only a minute sample is required for analysis, MALDI-TOF MS is particularly useful for identification of several fastidious and poorly growing species. Furthermore, studies have shown that the identification of anaerobic bacteria improves when the database of the MALDI-TOF MS is supplemented with reference spectra of species that are missing in the database or are underrepresented (<5 reference spectra) [12,14]. The pre-analytical factors which are required to obtain a good quality spectrum of anaerobic bacteria have also been assessed [6,10,13].…”