The hunt for the invisible
The Federal Institute for Occupational Safety and Health (BAuA) focuses on the development and application of new methods for the measurement of airborne bacteria at workplaces in a variety of projects.
In areas of work which have not been assigned to a protection level, risk assessment faces a difficult initial situation. In these cases, it can be beneficial to carry out workplace measurements in order to obtain data on the exposure situation. The first version of the Biological Agents Ordinance (BioStoffV) recommends determining the degree of air contamination at the workplace. Despite this, no legal measurement obligations apply, nor have any limit values been specified 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 site and target, both portable and stationary equipment are used. The separation of the samples is carried out with filters, on special surfaces, on solid nutrient media, and in collection liquids, depending on the collection system used. The materials or solutions are often varied within a particular system. 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 is almost exclusively realized using the cultivation-dependent approach. For the possible provision of proof using this approach, above all else, the biological collection efficiency of the collection system to be used (conservation efficiency) 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 nonculturable". To this day, the detailed causes of this remain unknown. "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 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 in use have long been 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 specificity for bioaerosol analyses.
Microorganisms are only rarely isolated from workplace samples and defined in further detail. However, this is a requirement for the acquisition of information as a part of 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 risk assessment, a closer identification of the microorganisms is therefore necessary.
To acquire information during measurement-based risk assessment, 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 obligatory 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 16S-rRNA gene’s information, and which can represent an uncertainty during the identification. This gene can occur within a cell multiple times, and partially show 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 bacterial communities in soil samples. For the targeted quantification of microorganisms or even species, with the use of specific primers, it is possible to determine the number of target genes in a sample using quantitative polymerase chain reaction (PCR) methods.
Regarding air analyses, especially when it comes to workplace measurements, 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 microorganisms, it is possible to take initial steps towards improved forecasting of the risk potential posed to employees and the environment, and to suggest appropriate protective measures.