Development of a MALDI-TOF database for the qualitative detection of bacteria in workplace samples

Status:

completed 03/2025

Aims:

To enable qualitative bacterial analyses to be performed by means of matrix assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF-MS), spectra of bacterial samples are compared with reference spectra from a MALDI spectral library. A library spectrum consists of a main spectrum profile (MSP), which is made up of numerous individual spectra of the respective bacterium. The library entries available at the present time have been built up primarily with bacteria that are of medical significance as potential pathogens; they do not therefore always include MSPs suitable for identifying bacteria sampled at workplaces.

Part 2 of the MALDI-TOF-MS project was therefore concerned with development of a procedure for establishing and populating a MALDI database at the IFA geared to workplace sampling, and making this database available to the German Social Accident Insurance Institutions within the framework of the MGU measurement system for exposure assessment. The advantage of an internal database lies in a higher identification rate in the institute’s laboratory, coupled with reduced outsourcing to external laboratories and greater independence from them, with associated savings in costs and time. The database is to be extended with bacteria, occurring in workplace samples (air or material samples), that cannot be identified by means of the existing MALDI libraries.

Activities/Methods:

Following on from MALDI project Part 1, IFA-2108, the procedure comprised the following steps:

1. Collection of bacterial colonies from workplace samples that could not be identified by means of the existing MALDI library (see IFA project 2108)
2. Molecular biological analysis of the isolates that could not be identified
3. Enrichment of the bacterium under a range of conditions
4. Performance of MALDI measurements and merging of all spectra into an MSP
5. A method was developed for validating the spectra entry against a bacterium of the same species but differing in origin, and was formulated in a work instruction.

Results:

To enable the IFA MALDI database to be built up incrementally, a work instruction was developed and used to produce 977 isolates that were not identifiable by means of MALDI-TOF. The work instruction describes cultivation of the bacteria. For example, two different culture media are used to produce high-quality MSPs. The database was structured to facilitate implementation of new entries whilst also permitting rapid searches in existing entries. The qualification of the MALDI-TOF MS and validation of the MSP required for generation of robust MSPs was also specified. This includes, for example, mass axis calibration, and testing of the identification performance of the MALDI-TOF by means of an E. coli standard. The creation of library entries is a complex task and must therefore always be performed by trained personnel.

The greatest challenge lay in clearly identifying the isolates selected for entry in the IFA-MALDI database. Molecular genetic sequencing was used for this purpose. Where identification by means of this method is not possible at the time of the analysis, the bacterial isolate is not used for the database. If, in future analyses, bacteria of the same species are identified within the pool of 977 selected isolates, they will be added to the existing MSP. This results in the library entry being geared increasingly precisely to the isolate. A software module for the internal laboratory software was developed for management of the isolates intended as MSP entries. The project thus led to creation not only of a work instruction, but also of the digital infrastructure required for documenting the analyses and results required for the database entries.

Last Update: