Microbiological investigation of cooling lubricants by cultivation dependent and cultivation independent methods

Status:

completed 03/2010

Aims:

- Development of standardized methods for the detection, quantification and identification of bacteria in metal working fluids.
- Especially detection of potentially pathogenic bacteria, i.e. mycobacteria.
- Applying of molecular methods to detect the bacterial diversity because of the restrictions of cultivation based methods.
- Using of cultivation based methods in addition in order to compare the results of both, cultivation-based, and cultivation independent approaches.
- Furthermore the development of a standardized procedure for the detection and quantification of mycobacteria from metal working fluids is one of the aims, in order to develop a rapid and reliable method as a basis of a risk assessment for workers exposed to metal working fluids.

Activities/Methods:

The current standard methods for the bacteriological investigation of metal working fluids are cultivation based, which means that the detection and identification of microorganisms depends on the cultivation on different agar-media. With this approach only those microorganisms can be detected, which are able to grow on the selected agar. A comprehensive statement about the overall microbial diversity in a sample is therefore not possible. Activities/methods of this project:
- Using of cultivation independent methods to describe the overall bacterial diversity in metal working fluids in more detail.
- Developing of a specific real time polymerase chain reaction (PCR) protocol for the detection and quantification of mycobacteria, which will be evaluated for standardized use.

Results:

The microbial diversity of ten metalworking fluid samples and seven samples of their water preparations was analysed. The samples originated from five companies, the oil for the water-based fluids originated from five metalworking concentrates.
In total, between 7.6 x 10⁴ and 1.6 x 10⁸ cells / ml (total cell count) and between 0 und 8.1 x 10⁸ CFU / ml (colony forming units; CASO medium) could be detected in the metalworking fluid samples. For the water preparations bases, between 4.6 x 10² and 7.8 x 10⁷ cells / ml (total cell count) and between 7.7 x 10¹ and 2.2 x 10⁵ CFU / ml (colony forming units; CASO medium) could be found.
70 isolates and 753 clones (cultivation independent analysis of the samples) were obtained and identified by 16S rRNA gene sequencing. 45 of the isolates were further analysed by physiological tests (assignment to genera and species). A high diversity could be detected in the metalworking fluid samples and in the water preparations bases. In total, 98 bacterial genera could be found by 16S rRNA gene sequencing.
Additionally, 21 genera could be detected by the identification on the basis of physiological test results. Only six of the species, identified by physiological test pattern, were found by 16S rRNA gene sequencing of isolates of the same sample, and only 13% of the genera, which were detected by cloning were also found among the isolatesl.
Because of these differences, several methods should be used in parallel for the diversity analyses of metalworking fluid samples.
20 isolates were found to be most closey related to Mycobacterium. They were obtained from mycobacteria-specific media. By cultivation independent analyses, no mycobacteria could be detected.
The number of different genera found in the metalworking fluids and the water preparations bases ranged between 3 and 20 genera per sample.
No congruence in the species compositions, detected in the metalworking fluid samples and their corresponding water preparations bases was found.
Of the five metalworking concentrates that were used (product A - E), the two products containing mineral oil with preconservation as basis, showed the highest microbial diversity. Of the two products that used mineral oil supplemented with Pseudomonas cells to prevent the growth of other bacteria, one product showed a high microbial diversity. In the other product, only 4 genera were detected. In product E, in which synthetic oil was used as basis, only five genera could be detected, with one dominating genus (Sphingomonas).
The majority of the detected bacteria, are considered as not hazardous. But some potentially pathogenic bacteria could be detected. Risk group 2 organisms (according to the TRBA 466) were e.g. some mycobacteria species as well as Citrobacter freundii, Brevundimonas diminuta, Aeromonas veronii biovar sobria and Pseudomonas alcaligenes. Control analyses of metal working fluids on a regular basis is regarded to be important for a risk assessement, that should be continuously updated.
A fast and quantitative standard method for the detection of mycobacteria was developed on the basis of a real time PCR protocol. With this method, the hsp gene of mycobacteria is amplified. This method worked for the majority of the samples, but further research is needed to use this method reliably for all metal working fluid samples.

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