Establishment of MALDI-TOF mass spectrometry for the qualitative detection of bacteria in workplace samples

Status:

completed

Aims:

For many years the Institute for Occupational Safety and Health’s (IFA) microbiology laboratory has performed qualitative bacterial analysis, exclusivelyusing biochemical tests (analytical profile index). The laboratory has years of experience in obtaining bacterial isolates from air and material samples of workplaces. These bacteria are not usually analyzed by external medical or environmental laboratories. In recent years, many microbiology laboratories have switched to using matrix assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF-MS) for bacterial analysis, a much faster and more powerful method. In this technique, bacteria are characterized by the spectrum of the bacterial proteome following protein ionization and mass spectrometric analysis. The IFA has followed this trend and procured a MALDI-TOF-MS system for its microbiology laboratory with the long-term aim of expanding bacterial analysis. The main advantages of MALDI-TOF analysis over traditional analysis methods are the automated spectral evaluation and the availability of measurement results within only a few seconds, enabling a large number of isolates to be analyzed within a short period of time. For that reason, experience is limited in the use of MALDI-TOF MS to identify bacterial species in workplaces. The aim of this project was to establish a standard MALDI method for the identification of bacteria to be used in the MGU (measurement system for exposure assessment)

Activities/Methods:

For the development of a standard procedure, samples from different workplaces were qualitatively analyzed for bacteria. These included samples from workplaces in areas such as anatomy, wallpaper and cardboard manufacturing, metal processing, car washes and photographer studios. The MALDI-TOF-MS-based bacterial analysis procedure generally works as follows:

1. cultivation of the bacterial consortium in the sample,
2. preparation of pure cultures of selected colonies for MALDI-TOF measurement,
3. analysis of the cultures by means of MALDI-TOF-MS.

The established physiological test methods also remained in use until 2022 for verification of the MALDI result. The methods were found to be too selective for verification purposes. Their specificity is advantageous for medical applications and is not suitable for screening applications. Where clear identification was not possible, the rRNA of the pure culture was used for performance of a molecular biological analysis, permitting subsequent clear characterization in MALDI-TOF (refer to Project 2109, MALDI database).

Results:

Two methods for identifying bacteria were developed during this project: the MALDI extended procedure (MALDI-ExtPro) and MALDI preliminary screening (MALDI-PS). The preferred method depends on the measurement task. With MALDI-ExtPro, analysis is performed on the purest possible bacterial colonies. Conversely, the MALDI-PS method is based entirely on the congruence of the analysis and database spectra. The MALDI-ExtPro method is generally more time-consuming (at least four weeks for cultivation + one week for quantification) than the MALDI-PS method. Any microorganisms that cannot be identified during the analysis are preserved and used later, to extend the IFA's MALDI database (refer to Project 2109, MALDI database). The project yielded two procedures by which the bacterial consortium can be analyzed from complex workplace samples by means of MALDI-TOF-MS, systematically and suitably for the measurement task.

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