Searching for the invisible
BAuA focuses on the development and application of new methods for the measurement of airborne bacteria at workplaces in a variety of projects.
Due to the difficult initial situation for a risk assessment in areas of work which have not been assigned to a protection level, the creation of a data basis for the stress situation can be beneficial to the completion of workplace measurements. The first version of the Biological Agents Ordinance recommends the determination of the degree of air contamination at the workplace. Despite this, no legal measurement obligations apply, nor have any limit values been specified and/or defined for biological agents at workplaces.
For the collection of airborne bacteria at workplaces, neither specific national nor international collection equipment - of which a variety of different commercial systems are currently available - have been prescribed for workplace measurements. Due to their separation principles, the measuring systems are grouped into six categories: filtration, impaction, sedimentation, precipitation (thermal and electrical precipitation) and impingement . Depending on the collection location and goal-setting, both portable and stationary equipment are used. The separation of the samples occurs with filters, on special surfaces, on solid nutrient media, and in collection liquids, depending on the collection system to be used. In this respect, the materials or solutions with a particular system are often used in the form of different variants. Therefore, an almost incalculable combination of collection systems and collection media, as described in the literature, is available. This means a comparison of the gathered data is only possible to a limited extent.
The detection of bacteria during workplace measurements works almost exclusively according to the cultivation-dependent approach. For the possible provision of proof using this approach, above all else, the biological collection efficiency (conservation efficiency) of the collection system to be used is considered decisive, as the bacteria experience different physical influences during their collection. The collection can lead to the death of the micro-organisms or the transition of the cells to a stage in which cultivation is no longer possible but the organisms remain alive. This stage is known as "viable but non-culturable". To this day, the detailed causes of this remain unknown. The "collection stress" also means that the collection periods have to be short. In the case of cultivation-dependent detection, collections over a full working shift should therefore be divided into appropriate, considerably shorter collection periods per collection.
It is also necessary to consider something else during cultivation-dependent analyses: due to the high metabolic diversity of the bacteria, it is to be expected that the selection of the nutrient media and incubation conditions means that it is only possible to detect bacteria, the cultivation requirements of which sufficient attention has been paid to. This phenomenon is known as the "plate-count-anomaly", and is based on the reduced measurement of micro-organisms via their cultivation in comparison with their direct microscopic counting. The previous exposure measurements and the measurements of emissions in organisations are primarily based on the detection of cultivation-dependent cumulative parameters like mesophilic bacteria. These provide the concentration as "colony-forming units per m³ of air".
To a partial extent, nutrient media are used, which aim to select groups of bacteria on a targeted basis. In the area of livestock stables, for example, selective media for the enrichment of staphylococci are frequently used.
Many of the "selective" nutrient media to be used are long established in the analysis of food or the area of clinical diagnostics. To enable a qualitative analysis of complex workplace samples, for most nutrient media, there is a lack of appropriate examinations which would highlight their selectivity and/or specificity for bioaerosol analyses.
Micro-organisms are only rarely isolated from workplace samples and defined in further detail. However, this is a requirement for the acquisition of information as part of a risk assessment. Therefore, the sum parameters to be detected can only act as general indicators, as it is not possible to derive a health- and/or environmentally damaging risk from them. Among others, this is attributable to the fact that both pathogenic and harmless species can be present within the detected groups of bacteria. For a risk assessment, a closer identification of the micro-organisms is therefore necessary.
To acquire information as part of a risk assessment which could be based on measurements, supplementary methods are required. The DNA sequence analysis of 16S-rRNA genes could be such a supplementary method. The clinical diagnosis already uses the sequence information for identification, and partially considers it to be the gold standard.
Identification via the analysis of the 16S-rRNA gene has a major advantage: at present, with every new description of a species of bacteria, it is necessary to save the corresponding sequence information in public databases. This means that large public databases such as the Ribosomal Database Project are available for the purpose of identification. However, with identification at the species level, other factors also play a role: It is also necessary to take the findings into account which, in recent years, have been gained surrounding the information of the 16S-rRNA gene, and which can represent an uncertainty during the identification. In this respect, this gene can occur within a cell multiple times, and partially have considerable intragenomical sequence differences. In addition, high sequence similarities frequently occur between the different types of a species.
The 16S-rRNA gene sequence analysis has been used for a long time, for instance, in the detection of bacteria community in soil samples. For the targeted quantification of micro-organisms or even species, with the use of specific primers, it is possible to determine the number of target genes in a sample using quantitative PCR methods.
In the area of air analyses and in the area of workplace measurements in particular, molecular biological methods have found little use. Further, the initial PCR-based analyses on the examination of bioaerosols occurred in the early 1990s and some of the studies clearly demonstrated that they were beneficial.
For this reason, in recent years, BAuA has completed projects which have addressed the development and application of new methods of workplace measurements. The results show that during the analysis of bioaerosols, molecular biological and biochemical methods detect the levels of exposure on both a qualitative and quantitative basis. In this respect, the tested methods represent an appropriate supplementation to the cultivation. Derived from the results and in connection with the risk groups of the detected micro-organisms, it is possible to derive initial steps for the improved forecasting of the risk potential posed to employees and the environment, and/or appropriate protective measures.