Identification of environmental Actinobacteria in buildings by means of chemotaxonomy, 16S rRNA sequencing, and MALDI-TOF MS.

Actinobacteria are abundant in soil and other environmental ecosystems and are also an important part of the human microbiota. Hence, they can also be detected in indoor environments and on building materials, where actinobacterial proliferation on damp materials can indicate moisture damage. The aim of this study was to evaluate the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of 28 environmental strains of Actinobacteria isolated from building materials and indoor and outdoor air samples, mainly collected in the context of moisture damage investigations in buildings in Finland. The 16S rRNA gene sequencing and chemotaxonomic analyses were performed, and results were compared with the MALDI-TOF MS Biotyper identification. Using 16S rRNA gene sequencing, all isolates were identified on the species or genus level and were representatives of Streptomyces, Nocardia, and Pseudonocardia genera. Based on MALDI-TOF MS analysis, initially, 11 isolates were identified as Streptomyces spp. and 1 as Nocardia carnea with a high identification score. After an upgrade in the MALDI-TOF MS in-house database and re-evaluation of mass spectra, 13 additional isolates were identified as Nocardia, Pseudonocardia, and Streptomyces. MALDI-TOF MS has the potential in environmental strain identification; however, the standard database needs to be considerably enriched by environmental Actinobacteria representatives. IMPORTANCE: The manuscript addresses the challenges in identifying environmental bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Biotyper-based protein profiling. The matter of the studies-actinobacterial strains-has been isolated mostly from building materials that originated from a confirmed moisture-damaged situation. Polyphasic taxonomy, 16S RNA gene sequencing, and MALDI-TOF mass spectrometry were applied for identification purposes. In this experimental paper, a few important facts are highlighted. First, Actinobacteria are abundant in the natural as well as built environment, and their identification on the species and genus levels is difficult and time-consuming. Second, MALDI-TOF MS is an effective tool for identifying bacterial environmental strains, and in parallel, continuous enrichment of the proteomics mass spectral databases is necessary for proper identification. Third, the chemical approach aids in the taxonomical inquiry of Actinobacteria environmental strains.

Toxicological and microbiological characterization of cow stable dust.

Exposure to farm environment has been shown to both protect from allergic diseases and increase the risk of respiratory syndromes. Mechanisms have been previously investigated by using farm dust extracts or specific components of dust. The use of authentic farm dust would better reflect the natural exposure. The aim of our study was to highlight the importance of proper assessment of the cow stable dust characteristics before conducting further investigations. For this purpose, we characterized microbiome and size distribution of unprocessed cow stable dust and its toxicological properties, as they have been often overlooked in search of protective factors. Stable dust samples from four Finnish dairy farms were collected by utilizing two different collection methods. Toxicological potential was analysed by stimulating co-cultures of lung epithelial and macrophage-like cells with dust. Size and mass distributions of airborne particles in the stables and bacterial and fungal microbiota of the dust were analysed. Stimulation with dust did not affect viability, but heightened oxidative stress responses and cytokine secretion, and slightly reduced the metabolic activity. There were a few differences in responses between farms, however, the differences were mainly in the intensity and not in the direction of the response. Cellular responses induced by dusts collected by different sampling methods did not differ substantially. Unprocessed stable dust samples showed relatively low direct toxicity but were able to trigger immune responses in studied cell model. This suggest that these dust collection methods could be utilized when investigating e.g. asthma-protective mechanisms.

Toxicity of airborne dust as an indicator of moisture problems in school buildings.

Moisture-damaged indoor environments are thought to increase the toxicity of indoor air particulate matter (PM), indicating that a toxicological assay could be used as a method for recognizing buildings with indoor air problems. We aimed to test if our approach of analyzing the toxicity of actively collected indoor air PM in vitro differentiates moisture-damaged from non-damaged school buildings. We collected active air samples with NIOSH Bioaerosol Cyclone Samplers from moisture-damaged (index) and non-damaged (reference) school buildings (4 + 4). The teachers and pupils of the schools were administered a symptom questionnaire. Five samples of two size fractions [Stage 1 (>1.9 µm) and Stage 2 (1-1.9 µm)] were collected from each school. Mouse RAW264.7 macrophages were exposed to the collected PM for 24 h and subsequently analyzed for changes in cell metabolic activity, production of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-6. The teachers working in the moisture-damaged schools reported respiratory symptoms such as cough (p = 0.01) and shortness of breath (p = 0.01) more often than teachers from reference schools. Toxicity of the PM sample as such did not differentiate index from reference building,s but the toxicity adjusted for the amount of the particles tended to be higher in moisture-damaged schools. Further development of the method will require identification of other confounding factors in addition to the necessity to adjust for differences in particle counts between samples.

Evaluation of sampling methods for toxicological testing of indoor air particulate matter.

There is a need for toxicity tests capable of recognizing indoor environments with compromised air quality, especially in the context of moisture damage. One of the key issues is sampling, which should both provide meaningful material for analyses and fulfill requirements imposed by practitioners using toxicity tests for health risk assessment. We aimed to evaluate different existing methods of sampling indoor particulate matter (PM) to develop a suitable sampling strategy for a toxicological assay. During three sampling campaigns in moisture-damaged and non-damaged school buildings, we evaluated one passive and three active sampling methods: the Settled Dust Box (SDB), the Button Aerosol Sampler, the Harvard Impactor and the National Institute for Occupational Safety and Health (NIOSH) Bioaerosol Cyclone Sampler. Mouse RAW264.7 macrophages were exposed to particle suspensions and cell metabolic activity (CMA), production of nitric oxide (NO) and tumor necrosis factor (TNFα) were determined after 24 h of exposure. The repeatability of the toxicological analyses was very good for all tested sampler types. Variability within the schools was found to be high especially between different classrooms in the moisture-damaged school. Passively collected settled dust and PM collected actively with the NIOSH Sampler (Stage 1) caused a clear response in exposed cells. The results suggested the higher relative immunotoxicological activity of dust from the moisture-damaged school. The NIOSH Sampler is a promising candidate for the collection of size-fractionated PM to be used in toxicity testing. The applicability of such sampling strategy in grading moisture damage severity in buildings needs to be developed further in a larger cohort of buildings.

The effect of assay type and sample matrix on detected cytokine concentrations in human blood serum and nasal lavage fluid.

Cytokine concentrations in biological fluids are widely used markers for activation of immunological processes. Confirming the reproducibility of measurements is important, especially in longitudinal or multicenter studies where time between analyses or different analyzing laboratories increases the intra-assay variability. In this study, the reproducibility of the cytokine analysis conducted with different assay platforms was studied by comparing the results of two cytokines [interleukin (IL)-6 in serum and nasal lavage fluid (NAL) and IL-8 in NAL] analyzed with Meso Scale Discovery (MSD) ultra-sensitive single and multiplex assay kits (n=76). In addition, the difference in cytokine levels between two biological sample matrices was studied by comparing the results of altogether 9 cytokines [IL-6, IL-2, IL-8, IL12p70, IL-1ß, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ, IL-10 and tumor necrosis factor (TNF)α] measured from serum and NAL of the same study subjects (n=460). The results show that the cytokine concentrations analyzed with single and multiplex assays are concordant but not equal. Comparison of the different matrices revealed that cytokine concentrations in serum do not correspond with concentrations detected in nasal lavage fluid. It can be concluded that comparability of the results from single and multiplex analysis of cytokines is high, but the concentrations should not be compared directly with each other. The differences between concentrations analyzed from serum and nasal lavage fluid indicate that the levels are specific for each matrix and represent distinct immunological conditions.