Effect of temperature conditions on the growth of Legionella species in complex biological systems for wastewater treatment

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Pathogenic Legionella spp., like Legionella pneumophila, can cause Legionnaire’s disease, a severe lung infection with a mortality of 5-10%. It is transmitted by the inhalation of contaminated aerosols from man-made water systems, such as wastewater treatment plants. Cases of Legionnaires’ disease increased in Europe by 65 % from 2015 to 2019. Legionnaires’ disease cases originating from wastewater treatment plants are expected to increase due to increased wastewater reuse applications and Climate Change. The latter in particular will increase wastewater temperatures, affecting also the microbial composition of wastewaters. To the best of our knowledge, a solution to prevent the multiplication of Legionella in wastewater treatment plants with warm wastewater temperatures (>23°C) is not available due to the lack of fundamental research. The objective of this proposal is to define the temperature conditions favouring the growth of pathogenic Legionella spp. from wastewater treatment plants, considering constant and dynamic temperature conditions.Isolates obtained from treated wastewater or activated sludge of five different wastewater treatment plants, treating industrial warm wastewaters, will be cultivated at five different temperatures between 20 °C and 40 °C. To test the effect of dynamic temperature conditions the temperature will be increased by 5 °C in a short time in the middle of the exponential growth phase. The Legionella’s growth parameters will be compared before and after the perturbation. A fast temperature increase of 5 °C was chosen according to our experience in monitoring Legionella spp. in wastewater treatment plants. The environmental Legionella isolates will be obtained, using the cultivation method. The affiliation of the environmental isolates and the Legionella spp. diversity in the biological stage will be analysed by genus-specific next-generation sequencing. Isolates for the temperature experiment will be selected covering the core Legionella community, present in all five wastewater treatment plants, and the unique phylotypes, only present in specific wastewater treatments plants.The integration of the results coming from the wastewater/wastewater treatment plant characterisation, the Legionella diversity and the temperature-dependent growth of Legionella environmental isolates from wastewater will advance our understanding of the role of wastewater treatment plants as an ecological niche for the growth of Legionella spp. . Our findings may be used to improve Legionella monitoring in wastewater treatment plants and to support the practitioners in the development of strategies to manage with sudden changes in temperature in wastewater treatment and reclamation facilities.