Diversity and resistance of microorganisms in a European spacecraft testing clean room

This thesis is part of ESA’s effort to advance Europe’s employed planetary protection policy to avoid unwanted contamination of our solar bodies due to mankind’s spaceflight program. Consequently the biological contamination of each spacecraft is monitored and, if necessary, reduced before launch. Therefore, the goal of this thesis was to identify and analyse the con-tamination level and phylogenetic diversity present in a European class 100,000 clean room (no more than 100k particles larger than 5 µm per foot3). In addition, the physiological properties of the cultivable bacteria collected from inside the facility were examined to identify possible changes in the bacterial characteristics due to the selective conditions present in such an environment. To achieve these goals three lines of work have been pursued: I. Evaluation and optimization of the established microbial sampling protocols Clean rooms represent a low biomass environment for which an effective and sensitive analysis of the collected samples is fundamental. The first step of this thesis was to critically evaluate existing microbial sampling protocols typically applied for the bioburden control of clean rooms. The swab technique, as specified by NASA (NPG: 5340.1D, 1980), was analysed by spiking an area with a defined number of spores and comparing the results of the swabbing method with the actual number of spores applied. During the analysis two major weaknesses in the sampling process were identified. First, only 50-60% of the actual spores present were picked up by the swab during sampling. Second, from those spores picked up, only 50% were again detachable from the swab and contribute to the final contamination count. These two factors will lead to an overall under-estimation of the real bioburden by up to 300% (Nellen et al., 2006). Once the analysis revealed that half of the collected microorganisms are retained in the swab, it was tested if this major shortcoming could be utilized. Several DNA extraction techniques were tested and combined to establish an effective method of directly isolating DNA from the swab-heads used during sampling. Specific protocols were developed, and it is now possible to identify cultivable and uncultivable bacteria from the same sample. The results of this optimization work will be incorporated into Europe’s future planetary protection bioburden monitoring protocols of space craft assembly facilities. II. Identification of the cultivable and uncultivable bacterial phylogenetic diversity of a class 100k clean room facility For this purpose ESA’s hardware testing facility (class 100k clean room) at ESTEC, (Noordwijk, Netherlands) was chosen as model system for a test sampling. After the sample collection, aerobic cultivation techniques and 16S rDNA based relatedness comparison were used in combination to identify the bacterial phylogentic variety present inside the facility. This work represents the first phylogenetic study of the cultivable and uncultivable bacterial fraction inside a European clean room. The integration of direct DNA analysis into the standard sampling assay raised the number of detected bacteria from 18 to 80 distinguishable species (≥97% 16S rDNA sequence similarity). Furthermore, it was possible to extract and identify the uncultivable bacterial fraction from each of the sampled locations; whereas, some of the locations did not yield any cultivable bacteria. This increase in phylogenetic data by molecular based methods is a known fact (Amann et al., 1995) and emphasizes the importance of including such methods into ESA’s bioburden monitoring process. Over 90% of the identified bacteria could be associated with three phyla, the Firmicutes (45%), the Actinobacteria (18.75%) and the Proteobacteria (30%). These 3 phyla are typical representatives of environmental and human associated samples and correspond to the two main contamination sources identified in this study: the human work crew (52%) and the urban surroundings outside the clean room (40%). Most of the detected bacterial species are harmless for the human work crew, nonetheless several pathogenic species were detected. These bacterial families should be monitored more closely during later studies. Only three species (Granulicatella adiacens, Mycobacterium chitae and Streptococcus mitis) could be identified in each of the three sampled access-restriction classes of the ESTEC facility. III. Analysis of the resistance potential of the collected cultivable bacteria Additionally, the question was addressed if the highly restrictive clean room environment could lead to a change in the physiological properties of the present bacterial community. The hypothesis was tested by using a microtiter based screening procedure to analyse the resistance potential of the cultivable bacterial community. The resistance characteristics of 62 collected isolates was tested against a selection of 23 harmful treatments. Though the difference in potency between different treatments (highly to mildly lethal) could be detected on the community level, no raised community resistance was observable under the tested conditions. It was striking that a high variance in the resistance characteristics between individual strains of the same species could be observed. Additionally, during this study several strains were identified which exhibit a marked increase in their resistance against one or more of the tested influences. These results lead to the conclusion that though changes in individual strains seemed to be promoted by the clean room conditions, no community level changes can be observed under the tested conditions. Furthermore, the molecular changes leading to the observed resistances need to be examined in detail to identify the source and if these resistances were obtained while the bacteria were inside the clean room or before they were brought into the facility.